Conference Agenda

Overview and details of the sessions of this conference. Please select a date or location to show only sessions at that day or location. Please select a single session for detailed view (with abstracts and downloads if available).

Session Overview
Date: Monday, 18/Mar/2019
10:00am - 10:30amWE: Welcome Session
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
Session Chair: Stephen Andrew Bustin, Anglia Ruskin University, United Kingdom
HS 14 
10:30am - 12:30pmNGB: Next Generation Biomarkers
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
HS 14 

Towards a Universal Diagnostic Test for Infection Using Rapid Nanopore Metagenomics

Justin O'Grady

Quadram Insitute Bioscience, United Kingdom

The rise in antimicrobial resistance (AMR) is predicted to cause 10 million deaths per year by 2050 unless steps are taken to prevent this looming crisis. Microbiological culture is the gold standard for the diagnosis of bacterial/fungal pathogens and antimicrobial resistance and takes 48 hours or longer. Hence, antibiotic prescriptions are rarely based on a definitive diagnosis and patients often receive inappropriate treatment. Rapid diagnostic tools are urgently required to guide appropriate antimicrobial therapy, thereby improving patient outcomes and slowing the development of AMR. In this talk, I will discuss the application of sequencing technology for the diagnosis of infection, focusing on rapid (~6hr) nanopore sequencing based clinical metagenomics.

MRNA As A Forensic Target - A Bridge Too Far?

Stephen Andrew Bustin

Anglia Ruskin University, United Kingdom

Individual human tissues are distinguished by distinctive RNA expression profiles, with the expression of many RNAs restricted to certain cell types only. Reverse transcription polymerase chain reaction (RT-PCR)-based assays are widely used in research and diagnostic applications to exploit this tissue-specificity for the identification of unique or aberrant cells. This feature has also led to an extensive evaluation of their potential to identify RNA transcripts that will reliably, sensitively and definitively identify tissues of origin in forensic casework applications. An analysis of the literature published by forensic scientists highlights a divergence of methodologies, protocols, reagents and interpretative models for forensic RNA studies. Disconcertingly, it also suggests the widespread application of protocols that are optimised for the analysis of DNA but are inappropriate for reliable RNA detection as well as the use of non-human and non-tissue-specific markers. Since the interpretation of RT-PCR-based results is dependent on the properties of the RNA as well as the variable characteristics of RT-PCR, this has serious consequences with regards to the robustness, consistency and reliability of any conclusions. A consensus on the most appropriate approach for reliable, accurate and meaningful RNA investigation as a forensic tool remains to be established.

HTPathwaySeq, a Novel Application for High-throughput RNA Sequencing Based Pathway Phenotyping

Pieter Mestdagh, Tom Maes, Manuel Luypaert, Nele Nys, Gert Van Peer, Ariane De Ganck, Carolina Fierro, Jan Hellemans, Jo Vandesompele

Biogazelle, Belgium

Different technologies support researchers in probing the transcriptome. The choice among these technologies is guided in part by the balance between the amount of data one wishes to obtain for a given sample and the number of samples being tested. Typically, these parameters are inversely correlated. At the opposite ends of this spectrum, deep RNA sequencing and qPCR yield in depth data for tens of samples starting at a total cost of at least 300€/sample or very directed information for thousands of samples at a cost below 10€ per sample, respectively. We here present HTPathwaySeq, a technology situated in the middle of this spectrum, tailored towards researchers looking for maximal molecular insights for their in vitrostudies.

At a cost below 100 EUR/sample, HTPathwaySeq processes 384 cell lysates with RNA seq to generate expression data analyzed at pathway level. Our data shows that shallow sequencing of crude cell lysates reproducibly detects over 5000 genes with at least 10 reads. Subsampling of deep sequencing datasets demonstrated that differential pathway analysis is largely unaffected when reducing the number of genes to this level. Consequently, reliable pathway insights can be obtained at high throughput and relatively low cost while not being limited to a predefined set of genes or pathways. In cell perturbation screenings (small molecules, RNAi, antisense or CRISPR), HTPathwaySeq can provide in depth information on the mode of action underlying the induced cellular phenotypes as well as molecular similarity scores to identify those perturbations acting similar to a reference condition or via shared molecular mechanisms. We will show results from a lead compound dose response study, illustrating the potential of HTPathwaySeq.

Expression Data Integration: Advancing Immuno-Oncology Target Discovery

Frank Staubli, Jana Sponarova, Stefan Bleuler, Philip Zimmermann

Nebion AG, Switzerland

Although genome-wide RNA expression analysis has become a routine tool in biomedical research, extracting valuable biological insight from thousands of published studies and underlying data remains a major challenge for two main reasons: the heterogeneity in annotations and technology, and the unreliable quality levels.

GENEVESTIGATOR is an analysis tool and database, containing manually curated gene expression data from public studies, making use of controlled vocabularies for several biological dimensions such as tissues, genotypes, diseases and treatments. To avoid bias in the results a strict quality control ensures to only integrate high quality samples and experiments into the database.

In this example study, we used GENEVESTIGATOR for 1. indication finding, 2. novel target predictions and 3. target validation for cancer immunotherapy as follows:

  1. Profiled the expression of selected immune checkpoint surface molecules across hundreds of tumors and subtypes to indicate which cancer types could be suitable for similar immunotherapies.
  2. Identified novel target genes that are co-regulated with known immune checkpoint targets across immune-oncology studies, for insights to which immunological pathways are affected.
  3. Compared spatial expression profiles of targets, to validate their combinative advantage.

These studies show how GENEVESTIGATOR can effectively take advantage of the world’s high-quality expression data, and help identifying new targets and characterize expression patterns of targets across cancers.

12:30pm - 2:00pmLU1: Lunch Monday
2:00pm - 6:00pmSC&MG: Single-Cell & Micro-Genomics
Session Chair: Mikael Kubista, TATAA Biocenter AB, Sweden
Session Chair: David Ruff, Mission Bio, Inc., United States of America
HS 14 

Spatial Transcriptomics - Bridging Histology and RNA Sequencing

Michaela Asp

KTH Royal Institute of Technology,Science for Life Laboratory,, Sweden

Spatially resolved transcriptomics provides us with new insights into the molecular diversity of heterogeneous tissue structures. Several approaches have been established in order to preserve gene expression information together with its tissue localization. However, existing challenges for many spatial technologies include the extent of existing knowledge about the targets, the labor-intensive nature of the methods or the fact that they are not applicable to clinical samples. Here, we present a method whereby whole intact tissue sections can be studied in a spatial whole-transcriptome manner.

Spatial Analysis Of Transcriptome And Proteome During Early Development

Radek Sindelka, Pavel Abaffy, Ravindra Naraine, Mikael Kubista

Institute of Biotechnology, Czech Republic

Starting from a single fertilized egg and then followed by various divisional steps, a complex organism is developed that has a distinct head-tail (bottom-up), left-right and dorsal-ventral (back-belly) asymmetrical axes. One of the main challenges in developmental biology is to understand how and when these asymmetries are generated and how they are controlled. Xenopus laevis (African clawed frog) and Acipenser ruthenus (sturgeon) are ideal models for studies of early development because of their large eggs and embryos. We have developed a unique molecular tomography platform based on RT-qPCR, RNA-seq and iTraq UPLC-ESI-MS/MS to measure asymmetric localization of fate determining materials such as mRNAs, non-coding RNAs and proteins within the early developmental stages. Additionally, we have developed new tools and approaches for analyzing data originating from spatial transcriptomics. Herein we present the results from our work.

High-throughput Single-cell Targeted DNA Sequencing from Tumor and Metastatic Samples Reveal Spatial Resolution of Evolutionary Trajectory Routes to Clonal Propagation

David Ruff

Mission Bio, Inc., United States of America

Recent advancements in genomic analysis of tumors have revealed that cancer disease evolves by a reiterative process of somatic variation, clonal expansion and selection. Therefore, intra- and inter-tumor genomic heterogeneity has become a major area of investigation. While bulk NGS has contributed significantly to our understanding of cancer biology and genomics, the genetic heterogeneity of a tumor at the individual cellular level is masked with the average readout provided by a bulk measurement. Very high bulk sequence read depths are required to identify lower prevalence mutations. Even at these high read depths, confidence confirmation in events at the 1% range or less is a formidable challenge. Rare events and mutation co-occurrence within and across select population of cells are obscured with such average signals. Additionally, recent reports highlight some of the crucial issues in whole exome studies for false detection rates. In an effort to explore this biology at higher resolution, we conducted single-cell targeted DNA analysis with the Mission Bio Tapestri™ Platform using sectioned melanoma metastatic tissues. Leveraging proprietary droplet microfluidics, the workflow unlocks access to gDNA, enabling high coverage uniformity of ~90% and low ADO of ~10%. Up to 20,000 cells can be interrogated with catalog or custom amplicon panels for any solid tumor type. Here, we use the Tapestri Single-Cell DNA Tumor Hotspot Panel that targets 59 commonly-mutated genes across 244 amplicons. We report that an analysis of multiple spatially-separated samples obtained from distinct metastatic sites in subjects revealed unique genomic signatures mapping to each solitary sample. These datasets support a number of conclusions including: 1) our recently optimized universal nuclei extraction process removes cellular components that are known to be highly inhibitory to PCR amplification – in this case melanin from melanoma cells, 2) single-cell analysis correlates strongly with bulk sample analysis, enabling confident comparison with previously-acquired results, 3) rare subclones, present at 0.15%, were detected, which is critical when monitoring disease progression, 4) single-cell analysis unambiguously identified the clones in each sample, enabling the reconstruction of clonal phylogeny, 5) the different clonal lineage observed in distant metastatic tissues highlights complex disease progression, and 6) tumor purity was measured at the single-cell genetic level. In summary, single-cell analysis offers to overcome the limitations of bulk NGS and can provide unique insights into the cellular-level complexities of tumor heterogeneity and phylogenesis. Here, the use of the Tapestri Platform demonstrates the power of single-cell DNA sequencing for characterizing solid tumor tissue samples and understanding disease evolution.

Using Single-cell Transcriptomics To Decipher The Formation Of Blood Stem Cells.

Christophe Lancrin

EMBL, Italy

In adulthood, the continuous generation of blood cells relies on the existence of hematopoietic stem cells (HSC), which have the ability to self-renew and generate all blood cell types. Any pathology affecting these cells could lead to the development of serious diseases such as leukemia and anemia. HSC are formed during embryonic life from endothelial cells, building blocks of the vascular system, which is responsible for blood circulation in the body. This process is called endothelial to hematopoietic transition (EHT). Using single-cell transcriptomics, we are uncovering the different populations involved in the process of HSCs’ formation. We are also using this method to understand how transcription factors regulate this process essential for the continuous oxygenation of the body and the establishment of the immune system protecting us from pathogens.

Single-cell Genomics – Are We There Yet?

Malte Paulsen, Diana Ordonez, Bianka Baying, Dinko Pavlinic, Jonathan Landry, Vladimir Benes

EMBL, Germany, Flow Cytometry Core Facility, Genomics Core Facility

The advent of single cell sequencing technologies created a very productive and collaborative environment for genomics and flow cytometric methods in the last five years. Flow Cytometry by nature was in the past the best method to analyse quickly thousands, if not millions, of cells for their expression of proteins, peptides or analysing the metabolic state with single cell resolution. Sequencing technologies have closed the gaps improving the quality and general robustness of sequencing DNA and RNA from single cells. This finally let to the development of multiple different, easy to use approaches in single cell generation and subsequent sequencing methods. Single cell sorting by FACS or Fluidigm C1 capture paired with sensitive library preparation methods paved the way. Nowadays, complete kit-solutions from 10xGenomics and their likes have rendered the initial resistance – or better put the initial need for intense technical skill development - to enter this method field almost to zero. Automation of analysis processes and kit-based scientific methods are probably the biggest driver for the high pace science that we are currently enjoying, yet at the same time it is most probably also responsible for the increased appearance of methodically poorly designed studies. We see a steep increase in studies that involve to a significant level single cell sequencing data. What strikes us is the apparent complacency of how authors set up their experiments. Studies with poor methodology are not rare in scientific literature and sadly, to some extent repeat the qPCR story.

Tailoring scRNA-seq to Meet the Challenges of Primary Cytotoxic T-cells

Kristiyan Kanev1, Patrick Roelli1,3, Ming Wu1, Christine Wurmser2, Mauro Delorenzi3, Michael Pfaffl1, Dietmar Zehn1

1Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, 85354 Freising, Germany; 2Division of Animal Breeding, School of Life Sciences Weihenstephan, Technical University of Munich, 85354 Freising, Germany; 3BCF, Swiss Institute of Bioinformatics, University of Lausanne 1015 Lausanne, Switzerland

Cytotoxic T-cells (CTLs) are major players in the protective immunity against viral infections and cancer. Given this central role, CTLs are of key interest for the development of novel and improvement of existing immunotherapies. This requires deeper understanding of the factors and molecular programs guiding their differentiation and functionality. This can be obtained through Single-cell RNA sequencing, which allows for unbiased discovery of single-cell resolved gene expression networks, which can be further used to define new cell subpopulations in an unbiased way. To allow for comprehensive single-cell resolved analysis of CTLs, as well as to deal with their minute amounts of messenger RNA, we have systematically optimize a droplet-based method providing breadth of cellular profiling and a plate based method providing depth of cellular profiling. We have achieved significant improvement in their sensitivity, providing tools for the establishment of an atlas of single-cell defined CTL cell states which will contribute to development of better conceptual understanding and new therapeutic opportunities.

A Deep Dive Into Single Cell RNA Sequencing Data

Patrick Rölli1, Kristiyan Kanev1, Delorenzi Mauro2, Zehn Dietmar1

1TUM, Germany; 2SIB, Switzerland

In the past 5 years, the number of single cell RNA-sequencing projects is expanding exponentially. In 2016, The human cell atlas was created with the main goal of creating a map of all human cells in order to help the scientific community in their research. The complexity of cancer research is pushing us to dissect and understand tissue sub-populations as well as their respective immunotherapeutic treatments which are all about cells and which of their mechanisms have to be harnessed. Similarly, the exploration of the human brain is also focusing more and more on specific cells in very specific area of the brain.

This has led the field of single-cell study, more particularly single-cell RNA sequencing, to grow exponentially as well. The number of methods that are available today to produce sequencing data is staggering and choosing which one is best suited to your needs can be tricky.

Being able to compare those protocols is critical to make an informed decision. Most of the commercial products propose their own tools to produce count matrices from the raw data. Those platform dependent tools are specifically designed for their own products and can be difficult to tweak for custom designs or experiments.

In order to compare and improve existing methods, we propose dropSeqPipe. A pipeline specifically designed to provide relevant quality control about most available single-cell platforms. Based on the snakemake workflow, it focuses on reproducebility, ease of use and flexibility.

During this talk we will present the tools and it’s different use as well as some examples of how it has helped our lab to improve our own protocol.

2:00pm - 6:00pmNGS-DA: NGS & NGS Data Analysis
Session Chair: Jo Vandesompele, Ghent University & Biogazelle, Belgium
Session Chair: David Langenberger, ecSeq Bioinformatics GmbH, Germany
HS 15 

RNA Atlas: A Nucleotide Resolution Map Of The Human Transcriptome

Lucia Lorenzi1, Francisco Avila Cobos1, Hua-Sheng Chiu2, Robrecht Cannoodt1, Tine Goovaerts1, Thomas Birkballe Hansen3, Pieter-Jan Volders1, Steve Gross4, Tom Taghon1, Karim Vermaelen1, Ken Bracke1, Jeroen Galle1, Jorgen Kjems3, Tim De Meyer1, Gary Schroth4, Pavel Sumazin2, Jo Vandesompele1, Pieter Mestdagh1

1Ghent University, Belgium; 2Baylor College of Medicine, US; 3Aarhus University, Denmark; 4Illumina, US

Technological advances in RNA expression profiling revealed that the human genome is pervasively transcribed, generating an unexpectedly complex transcriptome consisting of various classes of RNA molecules and a huge isoform diversity. Many of these RNAs show high tissue specificity, with some being expressed in only one or few cell types. While numerous large-scale RNA-sequencing studies have been performed, samples involved are often complex tissues, masking transcripts expressed in low-frequent cell populations, and sequencing methods typically focus on one class of RNA transcripts.

We assembled the most comprehensive human transcriptome across an extensive cohort of human samples, consisting of 160 different normal cell types, 45 tissues and 93 cancer cell lines. For each sample, total RNA, poly-A RNA and small RNA libraries were generated and sequenced using Illumina technology, yielding a total of 65 billion reads. Transcriptome assemblies for mRNAs, lncRNAs, miRNAs and circRNAs were matched with chromatin state maps from the Roadmap Epigenomics Consortium to define stringent gene models for each RNA biotype. Count data from polyA and total RNA sequencing libraries were combined to reveal the polyadenylation status of each transcript in each sample. We identified a total of 50235 gene loci of which 19668 were novel. From these loci, 37140 circRNAs were expressed. While a small fraction of novel genes was predicted to have coding potential, the majority of novel genes were non-coding, single exonic, and highly enriched for non-polyadenylated transcripts. Interestingly, a subset of genes showed variable poly-adenylation status across samples, mainly driven by alternative isoform usage. Biological information content of each RNA biotype was assessed by evaluating RNA expression - sample ontology associations and complex tissue deconvolution. Furthermore, we exploited the availability of intron reads from the total-RNA sequencing data to assess the regulatory potential of miRNAs, lncRNAs and circRNAs at the transcriptional and post-transcriptional level. Taken together, the RNA atlas serves as a unique resource for further studies on the function, organization and regulation of the different layers of the human transcriptome.

Getting More out of RNA-Seq Data: Transcriptomic Analysis of Ischemic Stroke

Peter Androvic1,3, Lukas Valihrach1, Denisa Kirdajova2, Martin Valny2, Miroslava Anderova2, Mikael Kubista1

1Institute of Biotechnology CAS, Czech Republic; 2Institute of Experimental Medicine CAS, Czech Republic; 3Faculty of Science, Palacky University Olomouc, Czech Republic

In recent years, RNA sequencing has become a standard method for genome-wide transcriptional analysis. Despite the extensive informational content of RNA-Seq data, many studies limit their scope to differentially expressed genes and/or pathway enrichment analysis, leaving substantial part of information unexplored. Here, we present a comprehensive transcriptomic analysis of the ischemic stroke in young and aged animals. We assessed differential gene expression across injury status and age, performed detailed pathway analysis and unsupervised co-expression analysis, identifying modules of genes associated with the various response to injury. We complemented these results with estimation of cell-type proportion changes using computational deconvolution techniques and assayed our results with findings from previous studies of similar design and publicly available databases. By employing these simple, yet often underutilized analytical approaches we found disease signatures consistent with literature and extend these results with new findings. We show strikingly variable response of different cell types and specific cellular pathways between young and aged ischemic animals, particularly related to immune response. Together, these results paint a picture of ischemic stroke as a complex age-related disease and provide insights into interaction of age and stroke on cellular and molecular level.

Liquid Biopsies For Personalized Medicine: The Omiterc Project

Francesca Salvianti, Irene Mancini, Lisa Simi, Stefania Gelmini, Mario Pazzagli, Pamela Pinzani

University of Florence, Italy

OMITERC is a data-sharing project sponsored by Regione Toscana, Italy, with the objective to develop an electronic registry that aggregates and links cancer genomic and pharmacogenetics-pharmacogenomics data with clinical outcomes from wild-type BRAF metastatic melanomas and RAS mutated metastatic colorectal cancers. The project aims to aggregate, harmonize and share clinical and molecular data obtained during routine medical practice. To reach this goal the implementation of a comprehensive database including all the clinical and molecular data deriving from the analysis of the primary tumor and the liquid biopsy in the case study is ongoing.

Twenty RAS-mutated metastatic colorectal cancer patients and eleven BRAF-wild-type metastatic melanoma patients were enrolled in the study and submitted to serial blood sampling before therapy and at different time intervals during the follow up.

We present the data deriving from the analysis of the liquid biopsy before and during therapeutic treatment in metastatic colorectal cancer patients: circulating tumor cell (CTC) detection and counting by CellSearch and mutational analysis by targeted NGS of cell-free DNA (cfDNA) and single CTCs (in a subset of cases).

CTCs were detected in 7 patients at baseline and were not found in subsequent blood draws during the follow up except for 4 subjects. On the whole the presence of CTCs showed a prognostic significance and was correlated to the efficacy of treatment.

KRAS mutational status in cfDNA from colon cancer patients at baseline was concordant with that of the primary tissue in 90% of cases.

The longitudinal study of cfDNA allowed a dynamic monitoring of the disease through the assessment of the presence of specific tumor-related mutations and the evaluation of their allelic frequency over time.

From the results emerges that it is important to study both CTCs and cfDNA, since they represent two different aspects of the liquid biopsy that can be integrated into a non-invasive approach to cancer patients.

Tiled Amplicons Panels in NGS-Based Genetic Testing

David Langenberger

ecSeq Bioinformatics GmbH, Germany

Next-generation sequencing increasingly replaces traditional Sanger sequencing for routine genetic testing applications. The higher throughput allows higher sensitivities for detecting low-frequency DNA mutations. However, more sequence reads do not automatically lead to a higher sensitivity and accuracy. Technical limits grounded in NGS technology and the library preparation, such as PCR duplicates, low complexity and false positives, need to be addressed. This leads to an increase in the diversity and complexity of available commercial NGS sample preparation kits.

Recent kits targeting low-frequency variant detection (for oncology) combine approaches such a unique molecular identifiers, single primer extension and tiled amplicon designs to address these issues. We show how these approaches work, and what are the practical consequences of these approaches on the observed sequence reads and on the downstream bioinformatics analysis.

PCR Based Target Enrichment For Variant Confirmation, Gene Panels And Multiplex PCR Sample Tracking In A Whole Exome Sequencing Workflow

Frauke Coppieters1,2, Thalia Van Laethem3, Matthias De Smet1, Paul Coucke1,3, Kathleen Claes1,3, Elfride De Baere1,3, Björn Menten1,3, Jo Vandesompele2,3, Steve Lefever2,3

1Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium; 2pxlence bvba, Dendermonde, Belgium; 3Center for Medical Genetics Ghent, Ghent University, Ghent, Belgium


Targeted PCR-based resequencing is an important application in clinical diagnostics. Using our best-in-class primer design tool primerXL, we have designed almost one million PCR assays for both fresh frozen and formalin-fixed paraffin-embedded samples, covering the entire human exome. Over 6200 assays for hundreds of clinically relevant genes in total were wet-lab validated. In addition, over 5000 patient-specific variants, from exome sequencing, were confirmed using pxlence PCR assays. All singleplex PCR assays work under universal PCR conditions and result in equimolar sequencing coverage. As a latest addition, we present the compatibility of pxlence assays with multiplex PCR applications. As a first product, we designed and validated a cost-effective and flexible sample tracking test. This primer pool enables fast identification of sample swapping or contamination which may occur in laborious library preparation workflows.


Thirty SNPs were selected based on their minor allele frequency, exonic location and overlap with the capture region of exome enrichment kits. We evaluated three different mastermixes for multiplex PCR and two library preparation methods, followed by 150 bp paired-end sequencing on a MiSeq instrument (Illumina).


The SsoAdvanced PreAmp Supermix (Bio-Rad) resulted in superior homogenous coverage following multiplex PCR of all SNP assays (pxlence). No significant difference in coverage uniformity was observed between the Nextera DNA Flex and the NexteraXT DNA library prep method (both Illumina). In virtually all tested DNA samples (n=393), 86.29% of the SNPs had a uniform coverage within 2-fold of the mean. Based on the SNP genotypes, DNA samples could unambiguously be discriminated.


In conclusion, pxlence provides high-quality and versatile PCR assays for various targeted resequencing applications. Here, we designed and validated a novel sample tracking test for whole exome or whole genome sequencing, involving a straightforward single multiplex PCR reaction followed by DNA sequencing library prep. In principle, our strategy could also be used to design gene panel-specific sample tracking solutions.

Handling of Spurious Molecular Species Dictates the Outcome of High-throughput 16S rRNA Gene Amplicon Profiling.

Sandra Reitmeier

ZIEL Institute for Food and Health, Core Facility Microbiome/NGS, Technical University of Munich, Freising, Germany

16S rRNA gene sequencing has become a popular method for rapid and comprehensive analysis of the diversity and composition of complex microbial communities. However, this method is prone to technical artefacts at various levels of the workflow. The most common method to analyse 16S amplicon data is building a cluster of sequences, representing single microbial entities on a 97% sequence identity (OTUs). Diversity measures derived from OTU-based datasets are strongly influenced by parameter settings such as filtering of spurious OTUs. This is crucial because of interpretation, reproducibility and quality. This study aims to bring clarity about filtering thresholds, usable to exclude spurious OTUs from high-throughput 16S rRNA amplicon datasets.

To determine an appropriate filtering cutoff two types of mock communities are used: ten different communities from published studies and two in-house generated datasets. This was complemented by the analysis of fecal samples of four gnotobiotic mice, colonized with different mixtures of bacteria. To analyse the impact of filtering, two studies with open access to sequence datasets are used as reference.

By filtering data with the commonly used method of removing singletons, shows on average 71% of all OTUs to be artefacts. A filtering cutoff of 0.25% reduces the number to 1.17% in mock-communities and 3.57% in gnototiotic mice, while still capturing 85% of true positives. Even with a low cumulative abundance of 1.14%, these artefacts are appearing in the data set and are as well considered as sequences while building the phylogenetic tree. Especially richness is influenced by the absolute number of OTUs and shows different results in both reference studies (0.25% = 195 ± 78 and 156 ± 44; no singletons = 364 ± 140 and 531 ± 201). Intra-individual stability of the microbiome is dependent on the used filtering method as well as stability of richness which is less dynamic by filtering with 0.25% cutoff (p-value < 0.001). A shift of median unweighted UniFrac distance by 0.36 per individual can be observed, which assumes a more stable microbiome. It is noting that the outcome in first study is inversely for generalized UniFrac distance. The second study shows the same pattern for both methods and distances even though there is a difference in distances for unweighted UniFrac. This affects the interpretation of stability of the human gut microbiome.

With this work we would like to raise the awareness of interpreting the outcome of 16S r RNA gene sequencing data. Since there is no standardisation it is important to know the methods behind the analysis and to be sensitised about the possible impact of different filtering approaches and used distance matrices. Nevertheless, it is not only important to carefully interpreted results it is also important to obtain high-quality results. Using a proportional cutoff is an independent filtering method to remove spurious OTUs in microbial datasets.

QC Measurements for Predicting Downstream NGS Success with FFPE and Circulating Cell-Free DNA Plasma Samples

Christopher D'Jamoos

Promega Corporation, 2800 Woods Hollow Rd. Madison, WI

Quantity and quality of DNA from formalin fixed, paraffin embedded (FFPE) tumor tissue samples is highly variable, with degradation and crosslinking due to the fixation process leading to issues with amplification and difficulty in NGS analysis. An alternative to FFPE is circulating cell-free DNA (ccfDNA) from plasma or other biological fluids. Compared to gDNA, ccfDNA yields are typically low, with tumor cell present at significantly lower frequencies. Due to the inherent variability of FFPE and ccfDNA, knowing the quantity of DNA is not in itself reliably predictive of downstream NGS success. In this presentation, we describe novel methods for predicting sequencing result quality utilizing a multiplexed qPCR assay.

6:00pm - 10:00pmPO1: Monday Main Poster Session
All posters will be displayed in parallel at all three poster sessions PO1, PO2 & PO3 => the poster viewing can be done from "Monday Evening Main Poster Session" till "Wednesday Lunch Poster Session"
Lower Level 

Digital PCR To Quantify Evolution Of Mycobacterium Tuberculosis SNP Proportion During In Vitro Competitive Assay

Charlotte Genestet1, Cécile Jacot-des-Combes2, Elisabeth Hodille1,3, Fabiola Bastian2, Oana Dumitrescu1,3

1Centre International de Recherche en Infectiologie, INSERM U1111, Université de Lyon, Lyon, France; 2Plateforme DTAMB, CNRS, Université Lyon 1, Villeurbanne, France; 3Hospices Civils de Lyon, Institut des Agents Infectieux, Laboratoire de bactériologie, Lyon, France

Background: Tuberculosis caused by Mycobacterium tuberculosis (MTB) complex remains a deadly infectious diseases worldwide. MTB infection results in a variety of disease manifestations and epidemiological success: latent asymptomatic infection and/or progression to pulmonary or extra-pulmonary injuries, with an array of severity symptoms. To assess the performances of genetic variants, we used in vitro competitive assays with and without selection pressure and evolution of variant proportion was evaluated by digital PCR.

Method: Isogenic clones, differing by only one SNP in locus involved in the biosynthesis of lipids from the mycobacterial outer membrane, were mixed at various ratio to obtain increasing proportion of the mutation in the bacterial mixture. MTB mixture grew in liquid medium with or without rifampicin, a first line antibiotic, during 4 weeks; MTB DNA was extracted and proportion of mutation at the end of the experiment was evaluated by digital PCR.

Results: Without antibiotic pressure we did not obtained significant differences in growth curves and digital PCR did not reveal significant evolution of mutation proportion in the bacterial mixture. Conversely, under rifampicin pressure we observed a better growth of MTB with the increase of the mutation in the bacterial mixture and digital PCR revealed a significant selection of this SNP during rifampicin exposure.

Conclusion: This study highlights a selective advantage of the SNP studied during rifampicin exposure and validates digital PCR as a tool to explore MTB community dynamics in in vitro models.

Development of a Strain-specific Droplet Digital PCR (ddPCR) for Detection and Quantification of a Probiotic Strain Bifidobacterium animalis (BAN) in Feed.

Sergi Raurich, Michaela Mohnl, Viviana Klose, Gerd Schatzmayr, Silvia Fibi-Smetana

BIOMIN Research Center, Technopark 1A, 3430 Tulln, Austria

The World Health Organization (WHO) defines probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Nowadays, the use of probiotics in animal nutrition to provide health benefits is common and widely accepted.

The use of real-time PCR is very common in the food industry, given that a method was determined to remove target DNA from non-viable cells through optimized DNA extraction. Nevertheless, the presence of inhibitors in feed samples can compromise the results and efficiency of this molecular technique. It has been demonstrated that the Droplet Digital PCR (ddPCR) in comparison to real-time PCR has higher tolerance to PCR inhibitors. In this study, a strain-specific ddPCR assay was developed for identification and quantification of a probiotic strain Bifidobacterium animalis (BAN) when added to poultry feed.

Therefore, the following chemical and physical parameters were taken into account: the optimal annealing temperature, concentration of primers and probe, and number of PCR-cycles. Ranges from 56.6°C to 62.6°C, 100 nM to 1000 nM, and 40 and 50 cycles were investigated, respectively. Optimal parameters resulted to be 59°C, 500 nM primers and 400 nM probe concentration, and 50 cycles. The linearity of the optimized ddPCR assay was established from the undiluted DNA until 1:10000dilution. For result validation, actual ddPCR results were compared to calculated results based on initial DNA concentration. The relation between expected results and ddPCR results was linear (R2=1), the efficiency (E) of the method was 99,98%. To warrant unbiased and repeatable ddPCR assay runs, purified BAN DNA was used.

To finalize the development, the Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantification (LoQ) for the assay were determined using poultry feed free of BAN, and spiked with different concentrations from 2,0E+06 CFU BAN/g feed to 1,91E+0 CFU BAN/g. Results showed a LoB of 3,07E+02 copies BAN/g feed, a LoD of 5,42E+02 copies BAN/g feed and a LoQ of 1,57E+03 copies BAN/g feed.

This ddPCR method is a valid method for quality control of animal feed supplemented with probiotic bacteria, to ensure appropriate inclusion rate of the product.

Design of a Digital PCR Assay for the Simultaneous Quantification of 14 Genetically Modified Soybean Lines in a Single Reaction

Katharina Lührig, Maximilian Neugebauer, Florian Priller, Heike Ziebarth, Hans-Henno Dörries, Cordt Grönewald, Kornelia Berghof-Jäger

BIOTECON Diagnostics GmbH, Germany


The cultivation of genetically modified (GM) crops has increased steadily over the last years and their presence in food products is regulated in many countries. The European Union (EU) has set a labelling threshold for food products containing more than 0.9% of authorized genetically modified organisms (GMO) in Regulation 1829/2003. This threshold is based on the total concentration of GM-events per ingredient.

The most common method for GMO quantification is standard-curve based real-time PCR. However, this method is usually limited to a single GMO event per assay. Since the number of authorized GMOs in the food market is increasing, faster and cost-efficient detection methods need to be developed to make monitoring feasible. Digital PCR is a suitable alternative to real-time PCR which allows multiplexed GMO quantification without the need for a standard curve. An additional advantage is the higher tolerance towards inhibitors present in plant material.


A digital PCR assay for quantification of all 14 soybean events currently authorized in the EU in a single reaction was developed by BIOTECON Diagnostics. In this multiplex assay all 14 GM soybean lines are quantified simultaneously in the FAM channel by amplification of their event specific sequences. The reference gene lectin is quantified in the HEX channel and the total GM soybean content of a sample is expressed as ratio between the species-specific reference gene and the sequences of the GM-events.


The assay is able to precisely and reliably quantify GMO contents at the regulatory thresholds of 0.9% and 0.1% which are stated in EU Regulation 1829/2003 and the German “GMO-free” label specifications laid down by Lebensmittel ohne Gentechnik e.V. (VLOG), respectively. Also the overall GMO content can be quantified in a single reaction from mixtures of events since all GM events are detected in one channel. The assay did not amplify DNA from plant species other than soy and is applicable to various relevant sample matrices. This was shown by spiking food products with DNA extracted from GM positive certified reference materials. The assay robustly separates positive and negative droplets in the digital PCR with only a low amount of rain for all events.


We successfully achieved quantification of all 14 soybean events in a single reaction. Using this new digital PCR assay quantification of approved soy GMOs is faster and cheaper than individual quantification by real-time PCR. Our assay fulfills all criteria to be used for routine monitoring of GM soybeans in food products.

A Rapid, Simple, High-throughput Compatible Approach to Generating CRISPR/Cas9 Knock-out Cell Lines

Meiye Wu, Steven Okino, Gerald Ui

Reagent R&D, Life Sciences Group, Bio-Rad Laboratories,

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and its associated endonuclease Cas9 have emerged as a revolutionary genome engineering tool. Using CRISPR/Cas9 to induce mutations in protein encoding genomic DNA regions is a powerful method for studying protein function in the native cellular context. However, the existing workflows for generating CRISPR/Cas9 knock-out cell lines are cumbersome, labor intensive and comprise of arduous rounds of cloning, limiting dilutions, and surveyor assays performed on dozens if not hundreds of clones. In order for large scale CRISPR/Cas9 studies to become practical and accurate for the genomics community, a streamlined process must be developed and validated at both genomic and protein levels. Here, we report a novel workflow that leverages the power of Droplet Digital™ PCR (ddPCR™), high-resolution melt analysis (HRM), and western blot analysis to generate 3 knock-out cell lines in under 5 weeks.

The Good, the Bad and the Ugly: Selective single cell isolation

Sandra Lubos1, Nils Körber1, Heide Marie Resch1, Iris Augustin2, Stefan Niehren1

1MMI - Microscopic Single Cell Isolation, Germany; 2University of Applied Sciences, Weihenstephan-Triesdorf, Germany

The isolation of single cells is an essential pre-requisite for many research projects in pathology, oncology or forensics.
The MMI CellEctor is designed to facilitate selective isolation of single cells in suspensions.
This study demonstrates the development of the novel Shake Mode feature which now enables for the efficient but gentle uptake of living adherent cells. Thus, cells can be used for all kinds of downstream analysis as well as for further cultivation.
As an application example, we show single-cell mRNA profiling using CTCs from liquid biopsies.

A SMARTer Solution To Stranded Single-Cell RNA-Seq

Matthieu Pesant1, Suvarna Gandlur2, Nathalie Bolduc2, Simon Lee2, Christopher Hardy2, Ankita Das2, Magnolia Bostick2, Andrew Farmer2

1Takara Bio Europe, Saint-Germain-en-Laye, France; 2Takara Bio USA, Inc., Mountain View, CA 94043, USA

Our SMARTer® NGS reagent portfolio has long included highperformance, cutting-edge solutions for RNA sequencing (RNA-seq). With the growing need for low-input and single-cell NGS library prep solutions, we see that researchers recognize the value in revealing transcriptome profiles from damaged cells as well as noncoding information from extremely low cell numbers (1–1,000). While we have previously released several industry-leading products that push the limits of sensitivity and reproducibility in RNA-seq from ultra-low inputs as well as single cells (SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing and SMART-Seq HT Kit), they both generate transcriptome profiles from mRNA only. Oligo(dT) priming is a very efficient way to capture the transcriptome, with minimal uninformative reads (e.g., from rRNA contamination), but it does not provide a complete view of the transcriptome, as only the polyadenylated fraction can be captured. In addition, for oligo(dT)-primed cDNA synthesis to generate high-quality libraries, one needs to start with high-quality, intact RNA, which excludes the use of this technology with samples damaged or degraded due to the nature of the processing (e.g., FFPE samples) or method of isolation. Additionally, these earlier single-cell kits do not preserve strand-oforigin information. All of these factors motivated the development of the SMART-Seq Stranded Kit, which allows for generation of sequencing-ready, stranded Illumina® libraries directly from 1–1,000 sorted cells or an equivalent amount (10 pg–10 ng) of purified total RNA of any quality. This kit integrates an innovative technology, already incorporated in our SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian, which enables removal of ribosomal cDNA following cDNA synthesis, as opposed to direct removal of corresponding rRNA molecules prior to reverse transcription. Since cDNA synthesis in the SMART-Seq Stranded Kit relies on random priming, rRNA is also captured, and remov al of the resulting cDNA prior to sequencing is essential. The SMART-Seq Stranded Kit protocol can be completed within seven hours, and a convenient pooling option for inputs between one to ten cells facilitates greater ease-of-use by minimizing the number of samples being handled (Figure 1).

TATAA Alu Repeat qPCR Assays – a Tool for Contamination Control and Quality Assessment in Single Cell and Cell-Free DNA Analysis

Alexandra Bergman, Andrei Herdean, Robert Sjöback, Mikael Kubista

TATAA Biocenter, Sweden

Laboratory reagents, including PCR master mixes, primers and probes, are commonly contaminated with residual human genomic DNA (gDNA) from the manufacturing process. For conventional measurements, the contamination can typically be considered negligible, but poses a significant, if not detrimental, problem in nucleic acid analysis applications where low-abundant targets will be studied, such as single cell and rare mutation analysis.

To test the purity of reagents TATAA Biocenter has developed ultra-sensitive qPCR assays targeting human specific Alu repeats. Alu repeats are short sequences of about 300 bp dispersed across our genetic code, summing up to more than one million copies per genome. This corresponds to more than 10% of our hereditary material, rendering Alu repeats the most abundant sequence of the human genome. Therefore, the TATAA assays will reliably detect even minuscules amounts of human gDNA contamination.

The TATAA Alu assays are designed to target multiple copies to generate reliable results also for very low amount of human gDNA. Alu assays are available that produce different amplicon lengths (60, 135 and 187 bp), which makes it possible to assess also the length distribution and integrity of the gDNA present. For example, via analysis of a sample with TATAA ALU-60 and TATAA ALU-187 assays, the level and fraction of cell-free DNA (cfDNA) in liquid biopsy samples can be assessed.

The ALU quality control panel was in part developed for the CANCER-ID consortium, developing standard operating procedures for liquid biopsy, and as quality control tool for ring trials run in collaboration with the SPIDIA consortium standardizing the preanalytical workflow.

SureCellⓇ ATAC-Seq Library Prep Kit: A New Solution for Single-Cell ATAC-Seq using Bio-Rad’s Droplet Digital Technology

Ronald Lebofsky, Carolyn Reifsnyder

Bio-Rad Laboratories, Digital Biology Center, Pleasanton CA

Cellular states and the activation of gene expression are governed by functional interactions between transcription factors and DNA regulatory elements. scATAC-seq (single cell Assay for Transposase Accessible Chromatin usingsequencing) is a powerful tool to interrogate chromatin accessibility genome wide at single-cell resolution. To enable the widespread and facile application of this approach, we developed a scATAC-seq platform using Bio-Rad’s Droplet Digital technology. Here, we describe a cell capture strategy that enables processing of thousands of cells per experiment with cell utilization efficiencies greater than 50%. We also show improved sensitivity and signal to noise relative to a previously published gold standard. The method is a push button workflow that can be comfortably completed in less than a day with only five user steps. We apply this technology to profile the epigenomes of tens of thousands of single cells across normal hematopoiesis and in response to immunological stimuli to discover a large repertoire of lineage- and stimulus-specific non-coding regulatory elements and transcription factors. Altogether, we show that these rich data sets and experimental tools provide a new platform for discovering regulatory differences across cells within heterogeneous tissues enabling the discovery of cis- andtrans-acting effectors of cellular differentiation and disease.

Single-cell studies: Focus on reverse transcription

Daniel Žucha1, Peter Androvič1, Mikael Kubista2, Valihrach Lukáš1

1Laboratory of Gene Expression, Institute of Biotechnology CAS, Czech Republic; 2TATAA Biocenter, Sweden

In the field of gene expression research, the reverse transcription (RT) is often the initial enzymatic reaction when processing the samples. However, it is known that the reaction output is far from ideal, as it relies on the choice of priming strategy, template and reverse transcriptase used.

The aim of this study was to compare the sensitivity, accuracy, reproducibility and yield of 11 commercially available reverse transcriptases with varying origin, RNase H activity and price. In consequence, two selected RTases were tested for further practical parameters, conducted on high-throughput platform. All measurements were executed under two priming strategies – routine RT-qPCR mixture of primers or reduced oligo(dT) concentration common in single-cell RNA-Seq protocols. Both single-cell and 100-cell bulk reactions were investigated.

Despite each reverse transcriptase performed with great accuracy and reproducibility, the ability to capture rare transcripts varied considerably. Similarly, differences were observed in reaction yields, where SuperScript IV and Maxima H- enzymes performed with significant efficiency. Consequent measurements confirmed Maxima‘s level of performance in single-cell RT-qPCR experiment, such as higher reaction positivity rate, signal strength or cluster separation. Methodical options of RT priming were also remarked, reporting considerable differences between priming alternatives. In final, the position of PCR targeted sequence has a minor role in its detection efficiency. Still, targeting of RNA 3’end regions was found to be the most reliable option.

Our results provide thorough comparison of available reverse transcriptases for both routine RT-qPCR and RNA-Seq applications, but also discuss the impact of RT priming strategy or template abundancy on reaction outcome. The choice of better RTase makes a substantial change in the outcome of RNA experiment. Practicality of PCR primer design is associated. Thus, this work serves as a guideline for improvement in RNA focused research.

High-througput, full-length, single-cell RNA sequencing

François-Xavier Sicot1, Kazuo Tori2, Magnolia Bostick2, Shally Wang2, Andrew Farmer2

1Takara Bio Europe, Saint-Germain-en-Laye, France; 2Takara Bio USA, Inc., Mountain View, CA 94043, USA

Since the emergence of next-generation sequencing (NGS), the importance and demand for single-cell analyses have risen rapidly. Single-cell RNA-seq can be used to generate data for analysis of differential gene expression, alternative splicing, gene fusions, and so on, allowing for cell profiling in detail. As a result, this technology has been gaining prominence not only in basic research fields, but also in the clinical space. Therefore, automation systems that can process many single cells regardless of cell type or size, and that provide high-quality data with high sensitivity, reproducibility, and fulllength sequence information are desired. Our method presented here for full-length RNA-sequencing the SMARTer™ ICELL8® automation system meets the demands described above. Our SMARTer ICELL8 system is an automated platform specialized for single-cell analysis. Unlike other automation systems, the SMARTer ICELL8 system utilizes a simple dilution method with a large aperture dispenser for single-cell isolation, enabling users to characterize many types of cells in a small amount of time. In addition, the SMARTer ICELL8 system provides the capacity to image and select cells for downstream analysis on a well-by-well basis, such that >1700 single cells can be analyzed at once. Our method for full-length RNA-sequencing the SMARTer ICELL8 system will enable users to perform all reactions required for Illumina® NGS library preparation in the ICELL8 chip, including cDNA synthesis and PCR amplification, so that the output from the chip is a sequencing-ready library. The most important feature of our approach is that the output library contains full-length sequence information as opposed to only capturing RNA 3’ ends (data typically used for differential gene expression analysis). Along with incorporating SMART® template-switching technology to ensure production of high-quality cDNA, our method for full-length single-cell RNA-seq with the SMARTer ICELL8 system enables various analyses to be performed with high sensitivity and reproducibility.

Clinicаl Аpplicаtion of Next Generаtion Sequencing Technology Methods

Elham El Darazi

USEK, Lebanon (Lebanese Republic)

The humаn body cаn be regаrded аs а constаnt temperаture fermenter for intestinаl microbes. The rich nutrients in the intestines аre the culture medium for bаcteriа. In fаct, we only hаve 1% The genes аre from pаrents, аnd the remаining 99% аre from microbes in the externаl environment (Bolcа et аl., 2013). They аre the bаcteriа in our body аnd in the intestines. Аt present, there аre more thаn 2,000 kinds of intestinаl microorgаnisms identified, аccounting for 1-3% of our body weight. In terms of quаntity, intestinаl microbes аlso hаve аn аbsolute аdvаntаge. When they аre mаny, they cаn reаch 10 times of humаn cells, but in generаl, they аre аt leаst equivаlent to the number of humаn cells. The lаtest estimаte is аbout 1.36 times thаt of humаn cells (Bolcа et аl., 2013).

The Humаn heаlth is insepаrаble from microbes. These lаrge numbers of microbes thаt breed on our body or in the body hаve аlreаdy tаken our body аs their "coloniаl": when the microbes settled in our bodies, they begаn to do whаt а colonizer should do: build а house, Eаting, breeding, resisting foreign enemies or invаding other florа (Аbubucker et аl., 2012).

In fаct, while аdаpting, microbes аre constаntly chаnging their living environment. Their excretа or metаbolites not only аffect their own survivаl, but аlso chаnge the metаbolism of humаn cells аnd the expression of genes. Once the gut microbiotа is out of tune, we will аlso be involved in them аnd get involved in their disputes; then inflаmmаtion, obesity аnd even cаncer come one аfter аnother (Greenblum et аl., 2012).

Bаriаtric surgery is а surgicаl treаtment for obesity. This type of surgery hаs proven to be more effective in treаting obesity thаn lifestyle chаnges, dieting аnd drug weight loss. Weight loss surgery аchieves the effect of inducing weight loss by chаnging different pаrts of the gаstrointestinаl trаct.

NGS is аlso known аs high-throughput sequencing, including second-generаtion mаssively pаrаllel sequencing аnd three-generаtion single-molecule sequencing (Mаrdis, 2008). There аre still mаny prаcticаl problems in the routine аpplicаtion of NGS technology in clinicаl tests. The complexity of clinicаl specimens mаy result in too little pаthogen informаtion аnd dаtа loss. Pаthogen dаtа mаy be mixed in the normаl gut microbiotа аnd it is difficult to distinguish (Keller et аl., 2014).

There аre no different specimens yet. Uniform provisions for pre-sequencing processing аnd pаrаmeter setting; high-throughput sequencing will generаte lаrge аmounts of dаtа, requiring huge dаtа аnаlysis. If there is а lаck of locаl dаtаbаses, the cost аnd time of computing work is difficult to control; for gut microbiotа, sequencing of genomic dаtа; the relаtionship with it is still uncleаr. Аccess to genomic informаtion аlone is not sufficient to clаrify gut microbiotа аnd the process of its development. Аccurаte аnаlysis аnd dаtа mining аre needed to аchieve precise treаtment аnd intervention.

Full-length 16S rRNA Sequencing Combined with the Illumina Barcode Structure Allows a Deeper Insight into Strains Present in Stool Samples

Isabel Abellan-Schneyder, Ilias Lagkouvardos, Klaus Neuhaus

Core Facility Microbiome/NGS, ZIEL - Institute for Food and Health,Technische Universität München, Germany

Background: Based on their ubiquitous presence, evolutionary stability and changes in its variable sequences, genes of the small ribosomal subunit are the most commonly used phylogenetic marker. Nevertheless, 16S rRNA analyses are mostly performed by short amplicon sequencing for the ribosomal gene of the small subunit (SSU) and are thereby limited. An easy, cheap and user-friendly method for full-length SSU rRNA sequencing should, therefore, be developed.

Results: We described a simplified method for full-length SSU rRNA sequencing using the Illumina MiSeq platform. The method has been improved such that the standard Illumina 16S barcode primers can be used and only adaptor primers in addition, which are low in cost are needed. Moreover, the protocol is shortened and tested for a better reproducibility compared to past proposals.

Conclusion: By using this approach, deeper insights into species and strains present in an unknown sample are possible. The method allows an easier data evaluation and is cost efficient because it can be easily adapted by all laboratories that already sequence amplicons with Illumina’s standard protocol by just adding a limited amount of novel primer.

Simple Whole Genome Amplification Technique Developed on the Basis of SD DNA Polymerase

Andreas Kirsten, Konstantin Ignatov, Ferdinand Holzinger, Vladimir Kramarov, Sergey Kovalenko

Bioron, Germany

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number of genomic DNA; WGA provides the only possibility to work with single-cells genomes so far. There are a number of WGA methods that have been developed. Degenerate Oligonucleotide-Primed PCR (DOP-PCR) is one of the simplest, fast and inexpensive WGA techniques. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an Improved DOP-PCR with enhanced WGA performance (iDOP-PCR) based on a Bioron’s novel DNA polymerase with a strong strand-displacement activity (SD polymerase, Patent US 9,896,671).

Several commercially available WGA kits were compared with the newly developed Bioron’s iDOP-PCR One Step WGA Kit. The amplified wgaDNA libraries were evaluated by different ways including NGS data.

iDOP-PCR WGA provided comparable or better quality of the amplified DNA libraries compared to the other WGA methods tested, the workflow is more simple, user-friendly and not labor-intensive. Thus, the new technique based on SD-polymerase can be used as a simple and convenient alternative for the Single Cells WGA in research and clinical practice.

Tackling Contaminants In Skin Microbiome Research

Claudia Hülpüsch1, Thomas Nussbaumer1, Vera Schwierzeck1, Amedeo De Tomassi1, Matthias Reiger1, Claudia Traidl-Hoffmann1,2, Avidan Neumann1

1Institute of Environmental Medicine, UNIKA-T, Augsburg, Germany; 2ZIEL - Institute for Food & Health, Freising, Germany

The skin microbiome is of interest as several skin diseases as atopic eczema are associated with a dysbalance in the microbiome. However, skin microbiome analysis is error-prone due to a low amount of microbial DNA in skin samples which increase the risk of contamination.

This has been confirmed by next-generation sequencing of the 16S rRNA of a dilution series of a synthetic bacterial mock community consisting of seventeen different species. The lower the starting material, the more OTUs appeared in the analysis which were not included in the mock community. A major contamination source for the samples are DNA extraction kits. Therefore, we used different DNA extraction kits to obtain DNA of skin swabs taken from adjacent skin site. The 16S NGS results show that samples cluster by DNA extraction kit instead of study participant, emphasizing the impact of contaminants from DNA extraction kits on the result in skin microbiome research.

In order to analyze skin microbiome data in a more accurate way, especially to optimize with objective criteria the handling of contaminations,we have developed a computational platform, MicrobIEM. This tool for microbiome analysis has a graphical interface. MicrobIEM facilitates the identification and exclusion of contaminating OTUs and samples in a user-friendly way based both on objective criteria and prior information. Remaining samples can be selected dynamically from the given meta-information for further analysis including the calculation of alpha and beta-diversity measurements and the taxonomy. Advanced statistical methods are used to allow testing the robustness of correlations with the meta-information even with a low number of samples.

In summary, our results and bioinformatics computational platform would allow better handling of contaminations in skin microbiome analysis.

Microsatellite Instability Analysis and NGS with Fragmented Sample Types

Christopher D'Jamoos, Samantha Lewis, Henk Honing, Brad Hook, Curtis Knox, Katie Oostdik

Promega Corporation, United States of America

Introduction: A significant hurdle to using fragmented DNA for genomic studies is obtaining a sample of sufficient quantity and quality for rigorous downstream applications like NGS. Having effective tools to isolate, characterize, and analyze fragmented DNA containing samples, such as circulating cell free DNA (ccfDNA) and FFPE tissues, can prevent downstream failures, ultimately saving hours of work and precious samples. Here we present optimized methods for use with even highly fragmented DNA samples. Using this toolset, we demonstrate successful NGS and microsatellite instability (MSI) workflows using matched FFPE tissues and plasma samples. Methods: Plasma and FFPE tissue samples were obtained from three individuals with colorectal adenocarcinoma. DNA was isolated with Promega’s Maxwell® RSC Instrument using the Maxwell® RSC FFPE DNA Kit for FFPE tissues and the Maxwell® RSC Circulating DNA Kit with the large volume custom protocol for plasma. DNA was then quantified with the ProNex® DNA QC Assay. Following quantitation, MSI analysis and NGS library preparation using the TruSeq Custom Amplicon Low Input Kit from Illumina was performed. NGS libraries were checked for size and quantity and then sequenced on the MiSeq® System (Illumina). Results: Full MSI profiles were obtained from DNA obtained from both ccfDNA and FFPE samples from each individual. Following successful determination of MSI-status, NGS libraries were produced from each sample. Sequencing of these libraries produced mean amplicon read depth greater than 3000x and mean coverage uniformity greater than 90%. In addition to excellent sequencing quality metrics, variants in mismatch repair genes identified in FFPE samples were also detected in matched plasma samples. Conclusions: Proper molecular tools and assays are essential to success in exacting downstream applications like NGS and multiplex PCR. This work introduces streamlined methods for DNA isolation, library preparation, and multiplex microsatellite instability analysis from fragmented sample types and demonstrates their effective use with matched FFPE and ccfDNA samples.

RiboPOOL: An Affordable Custom/Ribosomal RNA Depletion Solution Against Any Species for RNA Sequencing

Catherine Goh1, Andrew Walsh1, Michaela Beitzinger1, Jonas Bertram1, Kristina Doering3, Iana Kim4, Stefan Kotschote2, Claus Kuhn4, Konrad Foerstner3, Michael Bonin1

1siTOOLs Biotech GmbH; 2IMGM Laboratories GmbH; 3University of Würzburg; 4University of Bayreuth

With the rising use of RNA Sequencing (RNA-Seq) with Next-Generation Sequencing (NGS), there is a growing demand for reagents that increase output and reliability of sequencing data. Ribosomal RNAs (rRNAs) occupy more than half of total RNA sequencing reads. For sensitive detection of scientifically relevant RNAs, rRNAs are removed either by physical depletion methods using rRNA-specific probes or by isolating relevant coding RNAs through poly-A-tail affinity enrichment. Current commercial rRNA removal kits are costly and limited to well-studied species. riboPOOLs developed by siTOOLs Biotech in partnership with IMGM Laboratories and scientists from University of Würzburg and Bayreuth, present an affordable and flexible solution that gives scientists the freedom to deplete rRNAs or other custom abundant RNAs from any species. Composed of high complexity pools of optimally-designed biotinylated DNA probes, riboPOOLs specifically associate with cytoplasmic and mitochondrial rRNAs, enabling their removal with magnetic streptavidin-coated beads. Human and mouse riboPOOLs were demonstrated to outperform current commercial rRNA depletion kits (Ribo-Zero, Illumina) in RNA-Seq experiments, depleting ~10% more rRNA while leaving other RNA intact. riboPOOLs have been made against a diverse array of organisms including Planarian, Silkworm, Drosophila, Arabidopsis and Bacteria. Furthermore, riboPOOLs can be tailored to deplete abundant tissue-specific mRNAs, such as globin in red blood cells, making it applicable for NGS-based clinical diagnostics.

Sample Quality Control of Long Read Sequencing and Low Input Libraries

Kyle Luttgeharm1, Bettina Strauch2, Samina Kaufmann2

1Agilent Technologies, USA; 2Agilent Technologies, Germany

Long-read sequencing and miniaturization of library preparations are becoming increasingly common as new next-generation sequencing workflows are developed. Traditional quality control methods do not provide the required sizing accuracy of DNA greater than 50kb or the sensitivity allowing for sample conservation during the quality control steps. The Femto Pulse System by Agilent works to streamline quality control by separating gDNA up to 165kb in as little as 70 minutes, down from the 16+ hours required for traditional agarose PFGE. While the unparalleled sensitivity of the Femto Pulse allows for preparation of low input NGS libraries prepared from cfDNA, RNA, and miniaturized traditional DNA NGS libraries. Quality control metrics such as the RNA Quality Number (RQN) and user defined Genomic Quality Number (GQN) aids in the determination of sample quality. This poster shows use of the Femto Pulse system in large gDNA separation and low input library preparation with subsequent analysis features highlighted.

A Guide for Sample Processing to Determine Novel Mitochondrial Genomes by Next-Generation Sequencing

Sarah Viola Emser1, Martin Hofer1, Daniela Allmer1, Ingeborg Klymiuk2, Eva Millesi3, Ralf Steinborn1

1University of Veterinary Medicine, Vienna, Austria; 2Core Facility Molecular Biology, Center for Medical Research, Medical University Graz, Graz, Austria; 3Department of Behavioural Biology, University of Vienna, Vienna, Austria

Recent studies suggest that the functional repertoire of the mitochondrion had been underestimated. The organelle contains its own genome, the circular mitochondrial DNA (mtDNA), and communicates by anterograde and retrograde signaling with the nucleus, thus mastering metabolism, energy production, cellular signaling and innate immunity. Particularly, the mitochondrial single-stranded 7S DNA [1] and the mitochondrial derived peptides [2] representing key features of mitochondrial physiology emerged as topics of mitochondrial research. Here, we describe a simple, efficient and highly reproducible lab protocol for next-generation sequencing (NGS) of novel mammalian mtDNAs.

Without mtDNA enrichment, NGS analysis of the total cellular DNA results in 0.9% mtDNA reads (preliminary data). One option to lower financial expenditure caused by sequencing unwanted sequences of nuclear origin is to deplete linear nuclear enzymatically. It is reported that digestion with the Plasmid-Safe ATP-dependent DNAse followed by multiple displacement amplification results in a mtDNA proportion of up to 30% [3]. Generally, this approach is hampered by the recent finding of extrachromosomal circular DNA (eccDNA) constituting a considerable amount of the circular fraction of cellular DNA [3]. It is currently unclear whether the limitation also extends to platelet DNA, a template type naturally depleted of nuclear DNA. Alternatively, immunoisolation by magnetic beads coupled to an antibody against the mitochondrial outer membrane-protein TOM22 offers one of the most promising ways to increase the proportion of mitochondrial reads (up to 99%,)[4]. A similar level of sequence purity can be achieved by long-range PCR amplification primed by back-to-back oligonucleotides, the state-of-the-art approach to identify (human) mitochondrial genomes by NGS technology [5].

Here we address the issue of designing universal mitochondrial back-to-back primers for de novo sequencing of mtDNA across mammalian phylogeny tocombine cost-efficiency of NGS analysis with a reduced time expenditure for primer design.


[1] T. J. Nicholls and M. Minczuk, “In D-loop: 40 years of mitochondrial 7S DNA,” Exp. Gerontol., vol. 56, pp. 175–181, 2014.

[2] L. J. Cobb et al., “Naturally occurring mitochondrial-derived peptides are age-dependent regulators of apoptosis, insulin sensitivity, and inflammatory markers.,” Aging, vol. 8, no. 4, pp. 1–14, 2016.

[3] J. Zhu, F. Zhang, M. Du, P. Zhang, S. Fu, and L. Wang, “Molecular characterization of cell-free eccDNAs in human plasma,” Sci. Rep., vol. 7, no. 1, pp. 1–11, 2017.

[4] W. Quispe-tintaya, R. R. White, V. N. Popov, J. Vijg, and Y. Alexander, “Fast mitochondrial DNA isolation from mammalian cells for next- generation sequencing,” Biotechniques, vol. 55, no. 3, pp. 133–136, 2015.

[5] S. Luo et al., “Biparental Inheritance of Mitochondrial DNA in Humans,” Proc. Natl. Acad. Sci., vol. 115, no. 51, pp. 13039–13044, 2018.

Real-time Genotyping PCR From Blood And Blood Plasma Samples

Moritz Welter1,2, Ramon Kranaster2

1Chair of Organic Chemistry / Cellular Chemistry, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany; 2myPOLS Biotec GmbH, Technologiezentrum Konstanz, Blarerstraße 56, 78462 Konstanz, Germany,

Blood and blood plasma specimen are known to contain some of the most prominent inhibitors for DNA polymerases. Thereupon most diagnostic applications are based on isolated and purified nucleic acids. This strongly requires isolation and purification of DNA from the blood samples and removal of inhibitory compounds such as hemoglobin and immunoglobulin. Only afterwards, the samples can be analyzed in the respective PCR test. The necessity of the isolation and purification step renders the analysis more cost, labour and time intensive, is error prone and hampers throughput. Noteworthy, this isolation and purification step is currently higher charged in the German health-care system than the PCR of the genotyping test itself.

Based on the vast experience of myPOLS Biotec in the field of DNA polymerase engineering we were able to develop a novel system that enables real-time qPCR reliable analysis directly from blood samples without the requirement of isolation and purification of the DNA. On the poster, we present data of reliable real-time qPCR-based genotyping analysis of direct from certified blood samples on Factor II and Factor V blood samples employing different TaqMan and SimpleProbe assays. myPOLS Biotec now provides kits including this PCR innovation as research-use-only kits ( Currently we are further advancing our developments and are planning to provide this innovation as SNP-specific in vitro diagnostic-Kits within the on-going year i.e., especially for Factor II, Factor V, lactose intolerance, HFE, and HLA-B27.

Additionally, we are currently developing advanced real-time PCRs directly from plasma samples for liquid biopsy in collaboration with the University of Konstanz and show first results for the detection of the cancer mutation BRAF V600E.

Considerations and Quality Controls when Analyzing Cell-free DNA

Gustav Johansson1,2,3, Daniel Andersson1, Stefan Filges1, Junrui Li1, Helena Kristiansson2,6, Tobias Österlund2,6, Andreas Muth4, Tony E. Godfrey5, Anders Ståhlberg1,2,6

1Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 413 90 Gothenburg, Sweden; 2Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden.; 3Respiratory Inflammation and Autoimmunity, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.; 4Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden; 5Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA; 6Department of Clinical Pathology and Genetics, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden.

Analysis of circulating cell-free DNA (cfDNA) is a promising biomarker in cancer, prenatal testing, transplantation medicine and beyond. Ultrasensitive technologies enable detection of low (< 0.1%) allele frequencies, a pre-requisite to fully utilize the potential of cfDNA for these applications. Reliable cfDNA analysis requires that the entire liquid biopsy workflow is carefully optimized. Here, we illustrate important considerations for cfDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, both DNA fragmentation and the target sequence is shown to affect cfDNA assay performance. Finally, we show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.

Analysis Of Cell-free Tumor DNA Using SiMSen-Seq

Helena Kristiansson1,2, Daniel Andersson3, Tobias Österlund1,2, Stefan Filges3, Gustav Johansson2,3,6, Junrui Li3, Melita Kaltak3, Åsa Torinsson Naluai4,5, Anders Ståhlberg1,2,3

1Department of Clinical Pathology and Genetics, Sahlgrenska University Hospital, 413 45 Gothenburg, Sweden.; 2Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden.; 3Sahlgrenska Cancer Center, Department of Laboratory Medicine, Sahlgrenska Academy at University of Gothenburg.; 4Department of Laboratory Medicine, Biobank Core Facility, University of Gothenburg.; 5Biobank West, Sahlgrenska University Hospital, Sweden.; 6Respiratory Inflammation and Autoimmunity, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.

Analysis of circulating cell-free tumor DNA (ctDNA) in liquid biopsies offers new means for early cancer detection, real-time monitoring of treatment efficiency, and the discovery of relapse. Despite its potential use, ctDNA remains challenging to detect and to quantify as it represents only a small fraction of total plasma circulating cell-free DNA. We have developed an ultrasensitive sequencing technology, SiMSen-Seq, that allows allele frequencies < 0.1% to be detected, using several kilobases of DNA. SiMSen-Seq is simple to perform, flexible in multiplexing and requires minimal DNA input. SiMSen-Seq allows detection of variant alleles with easy customization of library content and a protocol that can be implemented in any molecular biology laboratory. Here, we present how SiMSen-Seq can be implemented in a liquid biopsy workflow, including sample collection, cell-free DNA extraction, sequencing and finally bioinformatics to quantify extremely lowly prevalent disease-specific mutations.

Performance Evaluation of Novel Protocols for Small RNA Sequencing In Biofluids

Sarka Benesova1,2, Peter Androvic1,3, Mikael Kubista1,4, Lukas Valihrach1

1Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic; 2Faculty of Chemical Technology, University of Chemistry and Technology, Prague, Czech Republic; 3Faculty of Science, Palacký University, Olomouc, Czech Republic; 4TATAA Biocenter, Gothenburg, Sweden

Circulating cell-free microRNAs are promising candidates for minimally invasive clinical
biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Small
RNA-sequencing (sRNA-Seq) has become the method of choice for the discovery of novel
miRNA biomarkers due to its high sensitivity and specificity, wide dynamic range, capacity to
discover new miRNAs and quantify expression of miRNA isoforms. However, numerous
technical biases associated with the small RNA-Seq library preparation hamper accurate
and reliable quantification. Recently, several new commercial kits for small RNA library
preparation claiming enhanced performance have been launched. However, an independent
evaluation of their performance was missing. Here, we present a benchmarking study
comparing six commercial kits, covering all of the currently available technical approaches
for the small-RNA library preparation. These include standard ligation of two defined
(Lexogen, Norgen), randomized (BiooScientific) or UMI-containing (Qiagen) adapters as well
as novel bias reducing approaches based on poly-A tailing (Takara) and single-adapter
ligation and circularization (Somagenics). We have used standardized human plasma
sample and set of synthetic spike-in miRNAs to evaluate several performance parameters
with focus on the utility of the kits for microRNA profiling from biofluids (sensitivity, tendency
to generate artefacts, spectrum of detected isomiRs, accuracy, false-positivity, ligation and
PCR bias) as well as practical parameters relevant for general users (level of multiplexing,
laboriousness of the protocol, time-consumption). Thus, this work can serve as a
comprehensive guideline for the choice of small-RNA library prep kit based on users´

Taqman Advanced Mirna Cdna Synthesis Kit To Simultaneously Study Expression Of Mirna And Mrna From Serum Samples

Astrid Ferlinz

Thermo Fisher Scientific, United Kingdom

MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. The interplay between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA can be valuable in understanding molecular mechanisms that may potentially have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse transcribe and pre-amplify miRNA and mRNA from sample limiting serum research samples using TaqMan® Advanced miRNA cDNA synthesis Kit.

Taqman Advanced Mirna Cdna Synthesis Kit To Simultaneously Study Expression Of Mirna And Mrna From Serum Samples

New Developments in Nucleic Acid Sample Quality Control

Bettina Strauch1, Rainer Nitsche1, Christine Voigt2, Martin Judex1

1Agilent Technologies, Germany; 2Alacris Theranostics, Germany

Quality control (QC) of RNA and DNA samples is key for the success of any downstream experiment. Especially, Next Generation Sequencing (NGS) developed to a powerful tool in almost all genetic research and diagnostic areas. Since the downstream applications are often time-consuming and expensive, tight QC steps are required to avoid a “garbage in-garbage out” situation. The ideal QC solution is easy-to-use, economical and provides fast and unambiguous results also for very low concentrated samples. Nucleic acid quality assessment can be standardized using automated electrophoresis systems to ensure that samples are “fit for purpose”. This poster exhibits the latest developments in nucleic acid sample QC and gives application examples – from RNA to Cell-free DNA (cfDNA) - evaluated with an Agilent 4150 TapeStation system. Cell-free DNA (cfDNA), gain more and more importance in the context of cancer research. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. Dependent on preanalytical sample treatment or extraction method, cfDNA samples may contain high molecular weight DNA fragments e.g. genomic DNA contaminations. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. For the objective quality evaluation of gDNA and RNA, the quality scores DNA integrity number (DIN) for gDNA and the RNA integrity number equivalent (RINe)for RNA can be assessed providing numerical values from 1 (degraded) to 10 (intact) for classification of samples.

Plasma cfRNA Profiling using the NanoString nCounter® Low RNA Input Assay

Alan Huang, Christoph Konig

NanoString, United States of America


Liquid biopsy, as a blood-based diagnosis with the advantages of easy access, less invasion, and repeatability (for longitudinal follow-up), offers the opportunity for early diagnosis, disease follow-up, and therapeutic monitoring. One example of this is plasma cfRNA profiling to discover disease-associated gene signatures. We conducted feasibility tests of the NanoString Low RNA Input assay using re-designed low input primers to analyze the expression profiles of plasma cfRNA.

Feasibility Tests and Results:

1. Primer efficiency assays by synthetic oligo standards:

Synthetic Ultramar DNA oligos to target 50 genes in the NanoString RNA panel were purchased from IDT DNA. The synthetic oligos were pooled and diluted in a series of dilutions to make the standards. Low RNA Input nCounter assays on the standards showed the lowest detection limit to be 30 molecules without false amplifications from non-template control. The average efficiency of the 50 primer pairs was 114% (range: 97%-136%) based on the standard curve analysis on the synthetic oligo standards.

2. Good correlation of cfRNA profiling between nCounter low input assay and real-time qPCR:

cfRNAs from 4 healthy donors were isolated from 1 ml cell-free plasma with the Norgen Plasma/Serum RNA Purification Kit. cfRNAs were amplified with the NanoString Low RNA Input kit using re-designed primers for 7 pre-selected genes from the NanoString Pancancer Immune Profiling panel (PCIP). PCR products were hybridized to the PCIP panel. The same primers were also used in the real-time qPCR analysis. Fold changes of 4 cfRNA samples to the universal reference RNA between nCounter and real-time qPCR data correlated well (R2 = 0.94) with good linearity (slope = 0.97).

3. Cell-free RNA amount increases with aging:

We further profiled cfRNA from 10 healthy individuals with 40 pre-selected genes from the PCIP panel to include abundant cfRNA genes found in the pilot study, oncogenes, and PBMC-derived genes. We found there is a trend of higher nCounter counts in the elderly population, which is consistent with a previous study.


1. cfRNA profiling can be performed on the NanoString nCounter platform utilizing optimized primers and 14-18 cycles of PCR amplification,

2. LOD of nCounter Low RNA Input assay is 30 copies using synthetic oligo standards.

3. Good correlation was seen between NanoString cfRNA data and real-time qPCR, one of the gold standards for cfRNA analysis.

4. cfRNA analysis of a 10-healthy individual cohort found the cfRNA amount positively correlated with age, suggesting elderly people tends to have higher circulating cell-free RNA in the blood.

5. The new improved protocol for low input RNA makes the NanoString nCounter platform a cost-effective and multiplexable molecular research tool for liquid biopsies.

Detection and Analysis of Free Circulating Long non-coding RNAs and mRNAs in Colorectal Cancer Pantients.

Pavel Ostasov, Pavel Pitule

Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Czech Republic

RNA is not confined to individual cells, but as was discovered in recent years, it is also part of different types of extracellular vesicles secreted by cells including ectosomes, exosomes, oncosomes and others.

Thus, RNAs isolated from these extracellular vesicles could be used as proxies to determine states of cells in various healthy or diseased tissues. While the focus is mainly on small circulating RNAs like miRNAs or piwi-RNAs, which are the most abundant species in these vesicles and outside of cells in general, we decided to focus on long circulating RNA species - long non-coding RNAs and mRNAs. While all RNA species allow analysis of their expression, long RNAs also allow detection of mutations, edits or splicing events. These might not only help to identify changes that are occurring during tumorogenesis but might also reveal mechanisms, that direct RNAs in these secretion vesicles.

In this work we analyzed publicly available sequencing data of exosomal RNAs1 as well as our data from extracellular vesicles derived from plasma of healthy volunteers and colorectal cancer patients. The analysis included presence of RNA regions, differential gene expression and splicing variants.

Overall, we detected changes in expression of some predicted splicing variants between healthy donors and colorectal cancer patients. Interestingly both publicly available and our own data contained a high levels of intronic sequences.

This work has been supported by the Charles University Research Fund (Progres Q39), by Charles University Research Centre program UNCE/MED/006 "University Center of Clinical and Experimental Liver Surgery" and by the National Sustainability Program I (NPU I) Nr. LO1503 provided by the Ministry of Education Youth and Sports of the Czech Republic.

1 Li, S., Li, Y., Chen, B., Zhao, J., Yu, S., Tang, Y., Huang, S. (2018). exoRBase: a database of circRNA, lncRNA and mRNA in human blood exosomes. Nucleic Acids Research, 46(D1), D106–D112.

Detecting Alzheimer’s Disease Risk Factors by qPCR Directly on Blood or Saliva Samples

Andrei Herdean, Mikael Kubista, Robert Sjöback

TATAA Biocenter, Sweden

Apolipoprotein E4 (ApoE4) is a major genetic risk factor for sporadic or late onset Alzheimer’s disease, with two single nucleotide polymorphisms (SNP) rs429358 and rs7412 respectively, being major predictors to the development of the disease (1). Furthermore, Alzheimer’s diagnosed patients that carry a specific allele of the BDNF rs6265 single nucleotide polymorphism have been shown to lose memory and thinking skills more rapidly than non-carriers (2).

TATAA Biocenter has developed three robust SNP genotyping assays for identification of the ApoE and BDNF alleles that predict development of late onset Alzheimer’s disease. We demonstrate the accuracy of our assays through analysis of know genomic DNA standards, synthetic DNA as well as benchmarking against other commercial SNP genotyping assays.

In conjunction with our new TATAA Direct qPCR GrandMaster Mix, we show that the step of DNA extraction from blood or saliva samples can be entirely removed from the SNP genotyping workflow. Here we present supporting data for performing qPCR SNP genotyping directly on blood or saliva samples, without the need for DNA extraction.


(1) Rasmussen, Katrine Laura. "Plasma levels of apolipoprotein E, APOE genotype and risk of dementia and ischemic heart disease: a review." Atherosclerosis 255 (2016): 145-155.

(2) Boots, Elizabeth A., et al. "BDNF Val66Met predicts cognitive decline in the Wisconsin Registry for Alzheimer's Prevention." Neurology 88.22 (2017): 2098-2106.

Building High Quality NGS DNA Libraries from Ultra-small Fragments

Stephan Bauer, Robert Brazas, Scott Monsma, Svetlana Jasinovica, Brandon Converse, Michael Lades

LGC, Biosearch Technologies, United States of America

Researchers are extending the use of next generation DNA sequencing and library preparation kits to highly challenging sample types such as ancient DNA and FFPE purified DNA as well as very small chromatin immunoprecipitated (ChIP) samples and cell-free DNA.In this study we describe a modified, modern library prep method for the construction of high quality DNA fragment libraries from extremely challenging and small samples. Examples for the application of this technology include both synthetic libraries using various size dsDNA oligos (10 bp to 90 bp) to demonstrate protocol capabilities as well as experimental data with cell-free DNA and micrococcal nuclease-digested chromatin immunoprecipitated material.

Liquid Profiling of Head and Neck Cancer: Circulating cell-free DNA in Plasma and Saliva for minimally invasive Cancer Monitoring

Romina Rösch1,2, Irina Kerle2, Markus Wirth3,6, Markus Nieberler4, Nicole Pfarr5, Carolin Mogler5, Silvia Thoene1,6, Ramona Secci1,6, Andreas Bietenbeck1, Florian Bassermann2,6, Jürgen Ruland1,6, Christof Winter1,6

1Department of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 2Department of Internal Medicine III - Hematooncology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 3Department of Otolaryngology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 4Department of Oral and Maxillofacial Surgery, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 5Department of General Pathology and Pathologcial Anatomy, Technische Universität München, Munich, Germany; 6German Cancer Consortium (DKTK), partner site Munich; and German Cancer Research Center (DKFZ), Heidelberg, Germany

In patients with head and neck squamous cell carcinoma (HNSCC), disease monitoring and detection of tumor recurrence are currently based on clinical examination, histological tissue analysis, and clinical imaging. This, however, does not sufficiently detect short-term changes in tumor burden. Tumor-derived materials, such as circulating nucleic acids, are present in body fluids of patients with cancer. The study of blood and saliva on molecular tumor constituents (liquid profiling) could enable better tumor monitoring and improve the early detection of recurrence. The aim of this work is, therefore, to analyze to which extent circulating, cell-free tumor DNA (ctDNA) and HPV DNA can be detected and which sample material (plasma vs. saliva) is more suitable for liquid-based tumor profiling of HNSCC patients.

In 91 patients diagnosed with HNSCC, blood was prospectively collected before therapy and in the course of the disease. In 9 patients, mutations in the primary tumor were analyzed with next-generation panel sequencing (45 genes) and ctDNA was examined in plasma with individually designed mutation-specific digital droplet PCR (ddPCR) assays. Further, all patients’ samples were analyzed by two mutation-specific ddPCR assays to detect commonly described hotspot mutations in HNSCC. This allows quantification of ctDNA even without prior tumor sequencing.
In 50 patients with tumors in the oropharynx and oral cavity, cell-free DNA (cfDNA) was tested for HPV (E7 oncogene-specific for HPV16) in plasma.

On average, 23 mutations (range 10 to 52) were detected in the primary tumor tissue by panel sequencing. In 25% (23 out of 91), tumor DNA was detected in plasma with mutation-specific ddPCR assays, in 22% already in early tumor stages (stage I (9%); stage II (13%)). In the course of disease monitoring an increase in tumor load (progressive disease) could be detected in the liquid biopsies on average 5,4 months (range 2 weeks to 13 months) earlier than by clinical manifestation.
In p16 positive patients (n=16) cell-free HPV type 16-DNA was identified in plasma in 39% (6 of 16) and thus allowed to identify the HPV type. In 50% of the cases, this was possible already at stage II. Specific mutations were observed in 75% of saliva samples (3 out of 4) and HPV16 DNA in 25% of cases (1 out of 4) to date.

Cell-free tumor DNA detection in plasma with digital droplet PCR is a promising monitoring tool for cancer profiling in early as well as advanced stages (III/IV) and could improve the early detection of recurrence. Saliva samples seem to be better suited for the detection of ctDNA than plasma. In HPV-associated tumors, circulating HPV DNA could be an additional profiling marker for disease monitoring of HPV-related cancer.

Anesthesia and Its Impact on miRNA Profiles in Circulating Extracellular Vesicles During Cancer Surgery

Dominik Buschmann1, Florian Brandes2,3, Anja Lindemann3, Melanie Maerte2, Petra Ganschow4, Alexander Chouker2, Gustav Schelling2, Michael W. Pfaffl1, Marlene Reithmair3

1Division of Animal Physiology and Immunology, Technical University of Munich, Freising, Germany; 2Department of Anesthesiology, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany; 3Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany; 4Department of General, Visceral, and Transplant Surgery, Ludwig-Maximilians-University Munich, Munich, Germany

While surgical resection represents the best chance of survival for patients with solid tumors, counterintuitive evidence suggests that surgery itself might facilitate tumor progression and metastasis. Data gathered in experimental and retrospective cohort studies indicate that anesthetic drugs commonly used in cancer surgery might have a differential impact on tumor cell biology, host immune response to surgery and tumor progression. The use of volatile anesthetic gases (VAG) may favor tumor cell growth and metastasis, while total intravenous anesthesia (TIVA) seems to exert anti‑tumor effects. Extracellular vesicles (EVs) and associated miRNAs are key mediators of intercellular signaling and metastasis, but little is known about how anesthetic drugs might impact circulating EVs.

To address this knowledge gap, we studied serum EVs in a cohort of colorectal cancer surgery patients. EVs were sampled prior to anesthesia using VAG (sevoflurane, n=9) or TIVA (propofol, n=8) and after tumor resection. EV‑associated miRNA profiles were analyzed by Next-Generation Sequencing (NGS) to identify transcripts with anesthesia-related perioperative changes in expression levels. Potential biological effects of miRNAs with altered pre- and post-surgical concentrations were assessed via in silico pathway analysis. Additionally, EVs sampled from patients in both groups and at both time points were biologically characterized.

In patients anesthetizes with both sevoflurane and propofol, pre- and post-operative miRNA profiles differed significantly. While there was significant overlap in the perioperative miRNA response, we detected five and 36 miRNAs that were specifically influenced by sevoflurane and propofol, respectively. Pathway analysis suggested that propofol-responsive miRNAs might exert inhibitory effects on cancer cell proliferation and migration and increase apoptosis, whereas miRNAs affected by sevoflurane did not impact these pathways. While size and marker protein composition of EVs were not altered by either type of anesthesia, EV concentrations in both groups were reduced after surgery.

In conclusion, our NGS data seem to match observations from retrospective cohort studies and point to EV-associated miRNAs playing a role in mediating the differential outcomes of cancer surgery using VAG or TIVA.

Sequence-specific release of EV-associated RNAs

Marie Mosbach, Christian Preußer, Lee-Hsueh Hung, Albrecht Bindereif

Justus Liebig University of Giessen, Institute of Biochemistry, Germany

Within the last decade, extracellular vesicles (EVs) turned more and more into the focus, due to their important role in intercellular communication. Although it was known already that cells release vesicles to their environment during apoptosis (apoptotic bodies), also healthy cells massively release vesicles. These EVs are important factors for cell-cell communication, acting as vehicles of biological material between parental and recipient cells. EVs contain different classes of RNAs, such as mRNA, miRNAs and circRNAs. As shown for miRNA and circular RNAs, RNAs are selectively exported into vesicles [1-3]. However, the factors and mechanisms that contribute to this specificity remain elusive. For example, a so-called Exo-motif has been described for miRNAs, which, however, cannot be transferred to all RNA classes and for circRNAs a possible size-dependent export was suggested [1]. In addition, only a few putative protein factors involved in packaging have been described [2].

To identify RNA sequence elements acting as export signals for the selective release of certain RNA species into EVs, we designed a modified in vivo SELEX approach (Systematic Evolution of Ligands by Exponential Enrichment). We generated a random sequence pool (N40), which was transfected and expressed in HEK293 cells. Specifically, several expression constructs were used, based on either an RNA polymerase II or III promoters. EVs were isolated from transfected cells, followed by RNA isolation, library preparation, RNA-seq analysis and bioinformatic identification of enriched RNA motifs. Sequence and motif enrichment analyses are now in progress. This unbiased method should contribute to our understanding of how RNAs are specifically packaged into EVs.

References: [1] Preußer et al. 2018, J Extracell Vesicles.; [2] Villarroya-Beltri et al. 2013, Nat Commun., [3] Hinger et al. 2018, Cell Rep.

Analysis of Total RNA Yield and Purity Using Different Extracellular Vesicle Isolation Methods

Rebekka Van Hoof1,2, Karen Hollanders1, Sarah Deville1,3, Patrick Wagner4, Jef Hooyberghs1,2, Inge Nelissen1

1Sustainable Health, VITO (Flemish Institute for Technological Research), Mol, Belgium; 2Theoretical Physics, Hasselt University, Diepenbeek, Belgium; 3Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium; 4Laboratory for Soft Matter and Biophysics, University of Leuven, Leuven, Belgium

Minimally invasive liquid biopsies for diagnosis and monitoring of specific mutations in lung cancer struggle to achieve accurate results.1,2 An innovative approach is based on extracellular vesicles (EVs)—vesicles released in abundance by cancer cells in the bloodstream.3 RNA localized inside EVs is protected against degradation and, although highly fragmented, it contains mRNA fragments that reflect the mutational status of the parental cell.4 Therefore, EV-derived RNA (EV-RNA) has the potential to improve current liquid biopsy-based diagnostics in terms of sensitivity and accuracy. However, little is known about the effect of different EV isolation methods on the total vesicular RNA profile.5 In this study the EV-RNA yield and purity of three commonly used methods is compared: ultracentrifugation, size exclusion chromatography and a membrane affinity spin column-based method, each combined with the exoRNeasy kit for RNA extraction.

Up to fifty times more RNA is extracted from EVs with the spin column-based method compared to ultracentrifugation or size exclusion chromatography. The purity of RNA also differs greatly between the methods. After EV isolation by ultracentrifugation and size exclusion chromatography there is a clear fraction of ribosomal RNA whereas the spin column-based method co-isolates RNA complexes. These data show the need for a standardized method for RNA extraction from EVs for clinical diagnostic application to enable more reliable and sensitive mutation analysis.


1. Heitzer, E., Ulz, P. & Geigl, J. B. Circulating Tumor DNA as a Liquid Biopsy for Cancer. Clin. Chem. 61, 112–123 (2015).

2. Passiglia, F. et al. The diagnostic accuracy of circulating tumor DNA for the detection of EGFR-T790M mutation in NSCLC: a systematic review and meta-analysis. Sci. Rep. 8, 1–10 (2018).

3. El Andaloussi, S. et al. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12, 347–357 (2013).

4. Enderle, D. et al. Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method. PLoS. ONE 10(8): e0136133(2015).

5. Van Deun, J. et al. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J. Extracell. Vesicles 3: 24858 (2014).

Urinary Extracellular Vesicles: Unveiling the Most Appropriate Purification Method with a View to RNA Sequencing and Biomarker Profiling

Veronika Mussack1, Georg Wittmann2, Michael W Pfaffl1

1Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Freising, Germany; 2Department for Transfusion Medicine, Cell therapeutics and Haemostaseology, University Hospital LMU, Munich, Germany


Small extracellular vesicles (EVs) with a size ranging from 50 – 200 nm are released by cells into the extracellular space. By resembling both physiological and pathophysiological conditions of their parent cells, EVs are important mediators in intercellular communication. As such, they have already proven as putative biomarkers in various bodily fluids. Particularly urinary EVs, which could be sampled non-invasively, demonstrate enormous potential in early urogenital disease detection. Especially vesicle-enclosed microRNAs (miRNA), are in the focus of biomarker research based on their high stability and fundamental activity as post-transcriptional regulators. However, when using high-throughput profiling via small RNA sequencing, strong dependencies of the results on the chosen EV purification method have been already identified for blood samples, leading to low comparability and reproducibility. Thus, we aimed to identify the most appropriate urinary EV purification strategy and, moreover, to check for the congruency of obtained results.


Urine samples were collected from six healthy male volunteers in consistent compliance with the Declaration of Helsinki. After pre-clearing, five different urinary EV purification methods were applied based on different strategies: spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient. EV preparations were characterized by transmission electron microscopy. Purified EVs were further analyzed for size and concentration by fluorescence nanoparticle tracking analysis using urinary creatinine to normalize for individual differences. EV-specific and non-specific protein markers were assessed by Western blotting. Total RNA was isolated and quality controlled by capillary gel electrophoresis prior to small RNA library preparation. Generated cDNA was fractionated by gel electrophoresis for miRNA size selection. Small RNA sequencing was performed using HiSeq 2500 (Illumina). Generated reads were processed according to an in-house developed pipeline and further analyzed by the DESeq2 algorithm.


All applied urinary EV purification methods yielded enough EVs and RNA for subsequent small RNA sequencing, albeit significant differences could be observed. Moreover, Western blot analysis revealed substantial co-purification of uromodulin in some EV preparations going hand in hand with higher sequencing readcounts. Exploratory data analysis of DESeq2-normalized reads further pointed towards purification-dependent differences in miRNA profiles, emphasizing the need for the combined evaluation of biochemical properties, proteomics and transcriptomics.


Summed up, care should be taken when selecting urinary EV purification methods appropriate for downstream analyses and the initial research question. Moreover, proper EV characterization is indispensable to ensure comparability and reproducibility of generated results.

Validating Sensitive Workflows To Analyze RNAs in Tumor-derived Extracellular Vesicles in Serum and Urine Samples of NSCLC Patients

Vera Kloten1, Rita Lampignano1, Martin Neumann2, Nina Kessler3, Anna Babayan2, Klaus Pantel3, Thomas Krahn1, Thomas Schlange1

1Bayer AG, Germany; 2Qiagen, Germany; 3Department of Tumor Biology, Germany

Liquid biopsy is defined as molecular analysis of rare cells or cell-free nucleic acids circulating in blood or in other biofluids. The concept of liquid biopsy aims at closely monitoring the status of a disease or treatment efficacy in a simple, fast, cost efficient way and at any point in time with minimal risk and burden for the patient. A preferable approach to investigate “liquid” RNAs is to focus on circulating extracellular vesicle (EV)-cargo (like mRNA and miRNA) rather than on cell-free RNAs, as the former is well protected from RNase degradation and therefore, more stable. However, the volumes of available blood samples can be limiting for molecular analysis and “liquid” RNAs are normally present in low abundance. Therefore, detection and quantification of RNAs in the blood requires robust and highly sensitive technologies.

In this pilot study, we investigated the absolute expression of 10 EV-transcripts typically involved in cancer development and chemotherapy resistance in longitudinal serum samples of 12 non-small cell lung cancer (NSCLC) patients (age range 37-76 years) treated with cisplatin at the Department of Inner Medicine II, University of Ulm, between May 2011 and August 2012.

Blood samples were collected at baseline, after 3-6 months and after 9-12 months of chemotherapy from patients showing objective clinical response. EV-RNAs were isolated by sequentially processing obtained serum samples with miRCURY® exosome kit and miRNeasy kit (both Qiagen, Hilden, Germany). Afterwards, the status of selected EV-transcripts was investigated via ddPCR (Biorad, Milan, Italy). Furthermore, we compared commercially available kits for the extraction of EVmiRNA from serum (exoRNeasy S/P Midi Kit) and urine (miRCURY Exosome Cell/Urine/CSF Kit) as well as cfmiRNA from serum or urine (miRNeasy S/P advanced). Extraction technologies were compared by measuring miRNA quantity and recovery of the exogenous control cel-miR-39.

After start of chemotherapy, we observed a downregulation of at least 2-fold of the following potentially cancer-related EV-transcripts : PTEN, ERBB2, FOSL1, IL-8, MET, RPS27A, SF3B1 and of the following housekeeping EV-mRNAs: ACTB, HIST1H3H and HSPA1A, in all patients. In addition to EV-transcripts, we showed an increased concentration of EVmiRNAs in serum and urine of NSCLC patients. However, using cel-miR-39 as spiked-in control, high extraction variabilities in patient samples were shown.

In this project, we demonstrated dynamic changes of cancer-related EV-mRNA expression in serum samples of NSCLC patients during cisplatin treatment. In addition to EV-transcripts, EVmiRNAs in serum of NSCLC patients might be useful as valuable “liquid” RNA biomarker as well.

This work is supported by IMI JU & EFPIA (grand no. 115749, CANCER-ID). Samples from patients and healthy volunteers, respectively, were collected under signed informed consent.

Serum-Free Media Supplements Carry MiRNAs That Co-Purify With Extracellular Vesicles

Martin Auber, Dominik Fröhlich, Krämer-Albers Eva-Maria

Johannes Gutenberg University of Mainz, Germany

Numerous studies report the association of miRNAs with extracellular vesicles (EVs). In most cases, EVs were harvested from cell culture-conditioned media containing fetal bovine serum (FBS) or a defined media supplement as nutrient. Recently, Wei et al. (2016, PMID: 27503761) reported that miRNAs are co-isolating with EVs when harvested in media supplemented with FBS or vesicle-depleted FBS. To avoid serum-associated miRNA contamination, we performed RNA-Seq of EV-associated miRNAs derived from primary oligodendrocytes cultured under serum-free conditions and subsequently validated miRNAs by RT-qPCR including media and supplement controls. Intriguingly, several EV-associated miRNAs were robustly detected in un-conditioned media subjected to the EV-isolation protocol and the media supplements NS21 and B27, which are routinely used for neural cell culture. RNAse and detergent treatment of NS21 removed most but not all of the contaminating miRNAs. Detailed analysis of NS21-supplement by screening individual components excluded BSA as major source of the miRNA contamination and identified a single component as carrier of miRNAs. Media supplement lacking the miRNA-carrying components however appears to affect cell viability, indicating that deprival of this specific supplement is not a solution to get rid of contaminating miRNAs. Based on these findings, we designed a new RNA-Seq strategy including EV-samples collected under conditions of supplementation or supplement-deprivation as well as supplement only control.

Taken together, our study shows that a single component of defined media supplements may carry major contaminating miRNAs into EV-samples. Consequently, analysis of EV-RNA needs refined strategies including respective media controls.

Systematic Comparison of Extracellular Vesicles from Human Arterial and Venous Blood: Highly Identical microRNA Expression Indicates Equal Use for Biomarker Applications

Stefanie Hermann1, Dominik Buschmann1, Benedikt Kirchner1, Melanie Märte2, Florian Brandes2, Stefan Kotschote3, Michael Bonin3, Marlene Reithmair4, Gustav Schelling2, Michael W. Pfaffl1

1Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Germany; 2Department of Anesthesiology, University Hospital, Ludwig-Maximilians-University Munich, Germany; 3IMGM Laboratories GmbH, Planegg, Germany; 4Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Germany

Extracellular vesicles (EVs) are crucial vehicles for intercellular communication in cardiovascular disorders such as cardiac hypertrophy and cardiac remodeling. To increase analytical specificity, biomarker studies often favor EV-associated microRNAs (miRNAs). While blood for diagnostic or research purposes is usually sampled using peripheral venous access, arterial lines inserted for monitoring are more convenient and often preferred in critically ill patients undergoing intensive care unit therapy or major surgery. This hampers comparisons between different pathologies and studies. We therefore tested whether EV-associated miRNA profiles depend on sample site location. Our study population included 20 cardiac surgical patients in which blood was simultaneously drawn from the radial artery and from the internal jugular vein prior to surgery. EVs were isolated using a polymer-based precipitation method, and total RNA was subsequently extracted. We characterized EVs by Nanoparticle Tracking Analysis, Western blot analysis and transmission electron microscopy. After small RNA sequencing, differential expression analysis was performed (DESeq2). Applying stringent filtering criteria to our data, no significantly regulated miRNAs were detected. Using less stringent filtering, we detected a subset of four miRNAs (miR-223-3p, miR-379-5p, miR-493-5p, miR-542-3p) slightly upregulated in arterial samples. We quantified these miRNAs by RT-qPCR. However, only one transcript (miR-493-5p), showing the highest regulation (1.6-fold), could be validated. Both arterial and venous EVs had spherical morphologies and equal particle concentrations in serum. However, arterial EVs were marginally smaller. Common EV protein markers were detected independent of sample type. Our data indicate that arterial vs. venous blood sampling should not represent a likely confounder when studying differentially expressed EV miRNAs. Therefore, data from studies using arterial or venous EV samples are probably comparable, with the exception of specific pathologies (e.g. severe pulmonary disorders).

Grp78 Plays A Crucial Role In The Exosome-Promoted Survival Of Irradiated Squamous Head And Neck Cancer Cells

Michael Schneider, Lisa Mutschelknaus, Klaudia Winkler, Rosemarie Kell, Michael J. Atkinson, Simone Moertl

Helmholtz Zentrum München, Germany

Background: Exosomes are nanometer-sized extracellular vesicles with functions in intercellular communication. Recent findings demonstrate that exosomes derived from irradiated HNSCC cells confer a more radioresistant phenotype to recipient cancer cells*. Here, we investigate the mechanism how exosomes promote survival in the radiation response of the head and neck cancer cell line BHY.

Methods & Results: Exosomes were isolated from conditioned medium of irradiated and non-irradiated cells by serial ultracentrifugation. Increased Grp78 was identified in exosomes from irradiated donor cells by whole proteome analysis** and by western blot. Moreover, we detected the localization of Grp78 at the exosome surface. In accordance to the increased Grp78 content of exosomes we also observed increased Grp78 expression in exosome donor cells (predominantly at the cell surface) after irradiation, while mRNA levels remained constant. We further observed increased surface expression of Grp78 in two other HNSCC cell lines; CAL-33 and FaDu. To test whether the exosomal Grp78 content can influence the Grp78 content of exosome recipient cells we cultured BHY cells with exosomes from irradiated and non-irradiated cells. We find enhanced Grp78 levels after incubation with exosomes from irradiated cells. To check a potential role of Grp78 in cell survival after irradiation we overexpressed the gene in BHY cells and performed a clonogenic survival experiment. Here we found that Grp78 alone enhances the radioresistant phenotype of BHY cells.

Discussion & Conclusion: Our results suggest a pivotal role of Grp78 transferred by exosomes in the radiation response of BHY cells. Radiation increases the cellular and exosomal Grp78 amount and moreover vesicle-mediated communication leads to enhanced Grp78 levels in exosome recipient cells. As Grp78 supports radioresistance, the transfer of this protein may contribute to the observed prosurvival effect of exosomes from irradiated donor cells. This study provides new mechanistic insights into the effects of exosomes in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy.

* Mutschelknaus L, Peters C, Winkler K, Yentrapalli R, Heider T, Atkinson MJ, Moertl S. Exosomes Derived from Squamous Head and Neck Cancer Promote Cell Survival after Ionizing Radiation.PLoS One. 2016 Mar 23;11(3)

** Mutschelknaus L, Azimzadeh O, Heider T, Winkler K, Vetter M, Kell R, Tapio S, Merl-Pham J, Huber SM, Edalat L, Radulović V, Anastasov N, Atkinson MJ, Moertl S. Radiation alters the cargo of exosomes released from squamous head and neck cancer cells to promote migration of recipient cells. Sci Rep. 2017 Sep 29;7(1):12423.

Analysis Of DNA Content From Human Melanoma Cellline Derived Extracellular Vesicles

Daniela Marie Brodesser1,2,3, Ingrid Walter4, Waltraud Tschulenk4, Sabine Brandt3, Gottfried Brem2, Franz Trautinger5, Joerg P. Burgstaller1,2

1Biotechnology in Animal Production, IFA Tulln, Austria; 2Institute of Animal Breeding and Genetics, Vetmeduni Vienna; 3Research Group Oncology, Equine Clinic, Vetmeduni Vienna; 4Institute of Pathology and Forensic Medicine, Vetmeduni Vienna; 5Department of Dermatology and Venereology, Karl Landsteiner University of Health Sciences


Extracellular vesicles (EVs) are a heterogenous population of membrane surrounded structures released by cells with varying cargo including nucleic acids, proteins and lipids. It is now established that these EVs have a significant role in cancer signaling. The nucleic acid (NA) content of EVs varies in amount and is both covering the nuclear (nDNA) and mitochondrial (mtDNA) genome. EVs are routinely extracted by differential centrifugation of cell culture supernatant, thereby separating enriched EV fractions (apoptotic bodies; microvesicles; exosomes). In this study, we characterized these fractions in three human melanoma cell lines; human fibroblasts and human melanocytes as controls. The EV-cargo was analyzed regarding their mtDNA: nDNA ratio. Furthermore, by using Proteinase and DNase treatments the surface-bound nucleic acids were digested to estimate the Distribution of DNA on the vesicular surface versus intravesicular DNA.


Melanoma Cell lines (A375, SKMel28, 518A2), human melanocytes and human fibroblasts were cultured in exosome depleted media for 48hrs; EVs were isolated with differential centrifugation of cell culture supernatant. A digestion step was performed using Proteinase K and Dnase followed by DNA extraction of each respective pellet enriched for apoptotic bodies, microvesicles and exosomes. MtDNA to nDNA ratio was quantified by qPCR (nuclear DNA: single copy gene MIA GenBank [NC_000019.10], multi-copy gene NPIPL (Sudmant, Kitzman et al. 2010) [NC_000016.10]; and mitochondrial DNA: MT-ND4 GenBank [NC_012920]).


Preliminary data indicates that the ratio of nDNA to mtDNA increases with each step in differential centrifugation. This suggests that in exosomes the relative amount of mtDNA is reduced compared to microvesicles, and even more in apoptotic bodies, irrespective of analyzed cellular origin. In an ongoing study the ratio of extravesicular vs intravesicular nucleic acids in the respective centrifugation pellets is analyzed.


The ratio of nDNA to mtDNA increases with each step in differential centrifugation, both in melanoma and fibroblast cell lines. Further research is needed to discriminate further between extravesicular vs intravesicular nucleic acids in the respective centrifugation pellets, and to elucidate the underlying mechanisms of nucleic acid distribution in EVs. This knowledge will be further transferred to the clinical level where tumour-derived EVs isolated from blood and the corresponding nucleic acids are going to be analyzed.


This work was supported by grant LS15-020 (to J.P.B.; F.T and S.B.) from Life Science Calls, Niederösterreichische Forschungs-und Bildungsgesellschaft (NFB;

P3py: A Python Module For The Multi-array MicroRNA Detection Data Analysis And The simulation Of Biomarker Diffusion And Reaction

Bo Zeng, Christiane Geithe, Stefan Rödiger

BTU Cottbus Senftenberg, Germany

MicroRNAs are small, conserved, noncoding RNAs regulating gene expression at the post-translational level, altered microRNA profiles have been implicated in many human diseases and thus used as clinical biomarkers. We developed a diffusion driven microbead multiple assay and combined it with an antibody based miRNA detection. The P3py python package was developed for the multiarray data analysis. P3py consists of two parts: microarray experimental data analysis and the computational simulation of microRNA diffusion and reaction. For the experimental data analysis, P3py can be used to preprocess microarray raw data, visualize fluorescence signal intensity using the 2D-space contour plot or heat map, statistical tests for the microbead distribution against the Complete Spatial Randomness (CSR), dose-response data curve fitting over time, fitted curve comparison under different experimental conditions. For the computational simulation, P3py solved the microRNA diffusion and reaction equations, and tried to predict the hybridized target concentration over time, there is a high correlation between the predicted and experimental hybrid target concentration.

Phenovault: An Open-Access Resource For Analysing RNAi And CRISPR Screens

Catherine Goh, Andrew Walsh, Michaela Beitzinger, Stefan Hannus, Michael Hannus

siTOOLs Biotech, Germany

RNAi and CRISPR screens give rise to a wealth of information on gene function. Recently published analysis focuses on a few top-scoring genes. Consequently, complete genome-scale screening data often lies buried within supplementary materials, invisible and untapped. The Phenovault is a growing database and analysis suite hosted by siTOOLs Biotech that contains complete, reagent sequence/ID-linked datasets from published RNAi/CRISPR screens. With over 20 million data-points, the Phenovault is the largest curated RNAi screening repository. Together with public and proprietary algorithms that harness the dominant microRNA seed-based behaviour of siRNAs, the Phenovault helps researchers uncover novel insights into their 1) siRNA reagents, 2) target genes and 3) RNAi screening datasets.

Optimal Use Of Statistical Methods To Validate Reference Gene Stability In Longitudinal Studies

Venkat Krishnan Sundaram1,2, Nirmal Kumar Sampathkumar1,2, Charbel Massaad1,2, Julien Grenier1,2

1Université Paris Descartes, France; 2INSERM U1124, Myelination and Nervous system pathologies - Group

Multiple statistical approaches have been proposed to validate reference genes in qPCR assays. However, conflicting results from these statistical methods pose a major hurdle in the choice of the best reference genes. Indeed, as their respective approaches to calculating reference gene stability is different, their suitability has to be tested for a given experimental setting. In this study, the stability of 10 candidate reference genes (Actb, Gapdh, Tbp, Sdha, Pgk1, Ppia, Rpl13a, Hsp60, Mrpl10, Rps26) was assessed using four common statistical approaches (GeNorm, NormFinder, Coefficient of Variation analysis and Pairwise ΔCt method) in a longitudinal setting. We used the development of the cerebellum and the spinal cord of mice as a model to assess the suitability of these statistical methods for reference gene validation. GeNorm and the Pairwise ΔCt were found to be ill suited due to a fundamental assumption in their stability calculations. Whereas, NormFinder and Coefficient of Variation analysis fare better provided they are used complementarily. We therefore devised a workflow combining these two methods for validating reference genes in developmental studies. This workflow proves to be more robust than any of the methods used individually.

A New Method to prepare DNA Libraries for NGS by using SD DNA Polymerase

Andreas Kirsten, Ferdinand Holzinger, Konstantin Ignatov, Vladimir Kramarov, Sergey Kovalenko

Bioron, Germany

Fragmentation of DNA is the first and very important step in preparing nucleic acids for NGS. Here we report a new method of NGS library construction based on a novel Fragmentation Through Polymerization (FTP) technique, which is simple, fast and user-friendly method. It generates double-stranded DNA fragments that are suitable for the direct use in NGS library construction, and allows eliminating the need of an additional step of reparation of DNA ends. The method allows simple and efficient addition of adaptors to the DNA fragments.

The FTP technique presumes the introduction of random nicks into the double stranded DNA by nuclease with simultaneous filling-in the DNA sticky ends which appeared as the result of nuclease action. Filling-in of the sticky ends requires strand displacement activity which is provided by Bioron’s SD polymerase, unique thermostable enzyme with strand displacement activity. The same enzyme can be used to amplify DNA fragments as soon as adapters are ligated to the ends of the fragment obtained.

In total, the procedure can be completed in 2-3 hours starting from genomic DNA and resulting in excess of PCR-fragments with adapters suitable for Illumina (or IonTorrent) sequencing.

BIOCEV GeneCore – More Than Just Gene Expression Profiling

Lucie Langerová, Filip Franko, Eva Rohlová, David Švec

Institute of Biotechnology CAS, Czech Republic

We present services of BIOCEV GeneCore - the best equipped core facility and service provider in the field of gene expression in Central Europe. We have broad experience in quality control (QC e.g. Fragment Analyser) in a single cell analysis (automated cell picking ALS Cellcelector), high-throughput and digital PCR (Fluidigm Biomark, BioRad QX200 Droplet Digital PCR System) and NGS library preparation.

We emphasise quality control, which is often neglected. Effective QC is based on the use of molecular tools to control contamination (RNA/DNA spikes), genomic background (ValidPrime) and quality of RNA (ΔAmp, RIN). We also take part in development of these methods to facilitate analysis of gene expression starting from bulk samples, down to the level of individual cells (direct lysis).

In addition to conventional qPCR analysis, we focus on single cells expression profiling and multi-analyte approach. Analysis of DNA/RNA/protein in parallel in one sample even on the single cell level provides comprehensive tool to map gene expression, to characterize types of cells, to determine the degree of differentiation and to study the pathological condition.

Currently, we provide assistance with library preparations and experimental design of RNA-Seq experiments, which are key preconditions for a successful project. Moreover, we offer new Two-Tailed PCR for ultrasensitive analysis of microRNAs based on an innovative novel design with a RT primer sensing the microRNA using two connected hemi-probes exceeding sensitivity and superior specificity is achieved.

MLH1 Promoter Hypermethylation: Development and Validation of a Methylation-Sensitive High-Resolution Melting (MS-HRM) Assay for use in a Lynch Syndrome Pre-Screen Pathway

Diana Pelka1,2, Sasha Hansel1, Ben Poskitt1, David Moore1,3, Tomasz K Wojdacz4,5, Phil Bennett1, Gareth Gerrard1,2

1Sarah Cannon Molecular Diagnostics, HCA Healthcare UK, London, UK; 2Faculty of Medicine, Imperial College London, London, UK; 3Department of Histopathology, UCLH NHS Foundation Trust, London, UK; 4MethylDetect ApS, Aalborg, Denmark; 5Pomeranian Medical University, Szczecin, Poland

Lynch Syndrome (LS) is associated with germline mutations in genes encoding the mismatch repair proteins, leading to mismatch repair deficiency (dMMR). dMMR can also arise somatically by promoter hypermethylation-mediated silencing of the MLH1gene. In colorectal cancer, there is a strong association between the BRAFVal600Glumutation and MLH1promoter hypermethylation, thus analysis of these markers, along with microsatellite instability (MSI), constitute components of the NICE-mandated LS pre-screen pathway. In the absence of a CE-IVD solution, we sought to develop and validate an MS-HRM based, MLH1promoter methylation assay for clinical use, using primers and control material from MethylDetect ApS, and bisulphite conversion and HRM-PCR mastermix kits from Qiagen.

Paired tumour/normal DNA was extracted from 20 FFPE samples: 12 colorectal, 4 prostate, 2 endometrial, 1 bladder, 1 unknown. Ten samples were MSI-High (MSI-H), 10 MSI-Stable (MSS); 5 were known dMMR, 2 MMR-normal; 4 were known BRAFVal600Glu, 10 BRAFWT; 1 from known LS, 3 from suspected LS. 20μL (5ng/μL) DNA was bisulphite converted (bsDNA) and 2-3μL used per HRM reaction. These were run on a Qiagen Rotor-Gene Q with 2-4 technical replicates for 45-50 cycles, with a melt start of 65-69oC and finish of 83-95oC. The paired-normal sample was used as the unmethylated baseline on a per-sample basis.

Repeated optimisation runs showed that 3μL bsDNA input, with 4 technical replicates, x50 cycles, and 69-83oC melt were the optimal parameters. All 11 MSS samples scored as unmethylated, as did the 4 known and suspected LS samples. The remaining 6 MSI-H samples (including 3/4 BRAFVal600Glu) scored as hypermethylated. MethylDetect control samples showed a limit-of-detection of 1% methylation.

All validation samples scored as expected, except for one BRAFVal600Glusample that scored unmethylated; this however came from a suspected LS patient and thus warrants further investigation. This assay proved to be rapid, cost-effective, sensitive, and specific.

Detection And Quantification Of MicroRNAs Using A Multiplex Microbead Assay To Accompany Quantitative PCR And Digital PCR Methods

Christiane Geithe1, Dirk Roggenbuck2, Katja Hanack3, Peter Schierack1, Stefan Rödiger1

1BTU Cottbus-Senftenberg, Chair of Multiparameter Diagnostics, Senftenberg, Germany; 2GA Generic Assays GmbH, Blankenfelde-Mahlow, Germany; 3University of Potsdam, Institute of Biochemistry and Biology, Professorship of Immunotechnology, Potsdam-Golm, Germany


MicroRNAs (miRNAs) are short (~ 22 nt) RNA molecules which act as post-transcriptional regulators on protein expression, cell differentiation, cell communication and immune defense. They can circulate into the bloodstream and other body fluids. MiRNAs are important clinical biomarkers for diseases, such as cancer, immune-related or cardiovascular diseases. Specific miRNA profiles e.g. from serum or plasma are promising tools for early in vitro diagnostics. Therefore, there is a high interest to develop sensitive, specific and robust methods for miRNA analysis. Quantitative PCR (qPCR), digital PCR (dPCR), microbead (MB) assays, microarrays and NGS are commonly used for the detection and quantification of miRNAs.

Our focus is the development of a non-invasive, multiplex MB-based detection system to accompany (quality control) the determination of specific circulating miRNA profiles from the serum of cardiomyopathy patients by qPCR and dPCR. An important aspect is to design the system in such a way that no amplification reaction is necessary.


Size/dye encoded MB were coupled via biotin-neutravidin bond with DNA capture probes which specifically targets the miRNAs of interest (miR-21, miR-197, miR-379, Let-7f). To achieve a direct comparability of the MB- and PCR-based methods, the miRNAs were first polyadenylated, afterwards hybridized to the capture probe loaded MB and detected via an ATTO 647N-labeled poly(T) sequence by using our in-house multispectral fluorescence imaging platform [1], which is based on a fully automated multispectral fluorescence microscope. For qPCR and dPCR, the polyadenylated miRNA was transcribed into cDNA and used in the same dilutions as in the MB assay.

Results and Conclusion

We developed a MB assay which enables the multiplex detection of so far four miRNAs from one sample in a concentration-dependent manner down to the lower nanomolar range. The comparison of the detection limit of the current MB assay with the quantitative PCR methods shows that these have at least 100 times higher sensitivity. The MB based miRNA detection is currently no more sensitive than the qPCR or dPCR. However, it offers some advantages regarding the multiplex degree as well as the amplification-free and isothermal reaction. Multiparametric analyses are relevant because they can work more cost-efficiently, require less sample material and more parameters can map disease patterns more accurately and thus make patient-related statements possible. Our assay has no amplification bias, as PCR based methods, since amplification can lead to a shift in certain target molecules and may serve as tool for miRNA sample quality control.

To increase the sensitivity of MB-based miRNA detection, we are working on the generation of poly(A)/(T) hybrid antibodies as well as miRNA-specific antibodies and the implementation of the assay into a microfluidic platform.

[1] Rödiger et al. (2013) Adv. Biochem. Eng. Biotechnol. DOI: 10.1007/10_2011_132

Developing a Customizable Panel of Real-time qPCR Assays on a Microfluidic Device for Respiratory Tract Pathogen Detection

Emmanuelle Lenotre

Thermo Fisher Scientific, United Kingdom

Respiratory tract infection is the most common infection in the world, presenting serious health issues to individuals and communities. A wide variety of bacteria and viruses are capable of causing upper and lower respiratory tract infections. Panel based testing using molecular methods to identify these pathogens has clear advantages over non-molecular or single target molecular testing.

A novel set of TaqMan® qPCR assays was developed to over 40 distinct respiratory pathogen targets, including bacteria, DNA viruses, RNA viruses, and fungi. These FAM™ dye-labeled assays were pre-loaded and lyophilized onto wells on TaqMan® Array Card™ (TAC), a microfluidic device that enables 8 samples tested for up to 48 targets simultaneously.

We report test data for sensitivity, specificity, accuracy and reproducibility of these respiratory tract pathogen assays on the microfluidic TAC.

The application enables researchers to study a large number of respiratory pathogens in a single reaction with a simple workflow, fast turnaround time, and high throughput yet flexible sample/target combinations.

Methods to determine Limit of Detection and Limit of Quantification of a quantitative real-time PCR (qPCR).

Amin Forootan

Multid Analyses AB, Sweden

Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

A Winter`s Week Tale in Sheffield Virology Department

Mehmet Onder Yavuz

Sheffield Teaching Hospitals NHS Trust, United Kingdom

Sheffield Teaching Hospitals NHS Foundation Trust (STH) is one of the UK’s biggest and most successful providers of hospital and community based healthcare. We provide a comprehensive range of local services to the residents of Sheffield, Yorkshire and Derbyshire. The virology department contains Serology, Molecular and Development sections employing 28 members of staff. We perform NA extractions in the Category 3 laboratory using the MagNaPure 96 or Qiacube extraction systems. We have dedicated rooms for each PCR stage for a unidirectional workflow. Weekly plan to cover all in PCR assays within stated turnaround times requires precise planning. ABI7500, Rotorgene Q, Panther, Veris and LC480 platforms to perform testing of Respiratory viruses, Enterovirus, Measles, Mumps, Norovirus, HSV, VZV, Atypical Pneumonia screen, MERS, P.jiroveci, BKV, EBV, HHV6, Parvovirus, CMV, HIV, HBV, HCV, CT, GC and T.vaginalis using qualitative or quantitative real time PCR. Our respiratory viruses’ panel contains 16 viruses covering Influenza, Parainfluenza, Respiratory Syncytial Virus, Human Metapneumo Virus, Corona virus, Rhinovirus and Adenovirus with DNA and RNA internal controls. During the Winter season there is a substantial increase in the number of respiratory samples tested. Commercial assays as well as in house developed assays are used for testing. All our in house assays have the same amplification profile enabling us to perform different PCR tests together and providing rapid results of urgent tests. Plasmid based positive controls as well as strict in house PCR batch approval systems ensure a consistent and efficient diagnostic procedure.

Quicker results can be obtained by:

  • Using Multiplex PCR. Our Multiplex Respiratory viruses’ PCR panel requires 6 wells for screening.
  • Increasing plate size for testing. Using 384 well plates instead of 96 wells plates has increase our capacity 4 fold.
  • Information technology packages such as Roche cITM or result transfer option from ABI7500 system help greatly for efficient, accurate and timely result processing.
  • Automation such as pipetting stations eliminate any pipetting errors and reduce hands on time .

Master mixes play a very important role for the development of and testing of PCR reactions. Highly stable and efficacious master mix will help producing consistent and correct results even if sample viral load is very low. The PerfeCTa qPCR tough mix and QScript custom XLT 1-step RT-qPCR toughmix are used for our in house PCR assays. Development of the assays was based on Taqman technology using the ABI7500 system. However, we are now in the process of revalidating our in house assays on the Roche Flow system using 384 well plates. I have chosen the week starting 7th January 2019 to highlight winter pressure on our Virology department and how we tried to overcome most of these issues. When compared to a week in summer there was a 4 fold increase in the respiratory samples received.

Effects Of The Use Of Degenerate Bases In Primers On Quantitative PCR Measurements

Laure Barbier, Catherine Rousseau, Charles Chapus, Thomas Poyot

IRBA, France

In our institute, we develop dual research programs to respond both to the armed forces aims and civilian uses. Different experiments are thus conducted in the field of NBC risks. To this aim, we studied the effects of degenerate bases incorporation on quantitative PCR measurements to improve diagnostic of biological agents presenting nucleotide polymorphism. Primers were thus modified using either inosine or 5-nitroindole, two “universal” bases. Different parameters were evaluated such as the type and number of substitutions, their location and their contiguity effects. Asymmetric PCR were also conducted in order to verify substitution’s effect on PCR stability. First, we designed forward and reverse primers containing 1 to 3 substitutions at different location of the primers. Then, we studied the effects of 6 substitutions placed on contiguity or not and on the first position of the 5’ end of the primer or not. All these primers were tested on synthetic matrices in term of PCR amplification efficiency and specificity of the reaction. For this, delta Ct difference and variation in melting temperature were calculated using amplification occurring without modified primers as control.

Our work revealed that nitroindole substitution led to a decrease in PCR performance whatever number of substitution on the 5’ or middle of the reverse primer. When one to three nitroindole were placed on the 3’ end of the primer, no amplification occurred.

When using inosine substitutions, we demonstrated that a single residue placed in the 3’ end of the primer had a negative effect on PCR performance. Such effect was potentiated with the increase in number of substitutions. When 6 inosine substitutions were present, inosine residue on the first 5’ end of the primer appeared essential. Moreover, PCR amplification was less destabilized when groups of 2 consecutive inosine were present on the primers.

Finally, this study demonstrated that, in our model, a better amplification occurred when residues were substituted by inosine instead of nitroindole. We also showed that bases modifications by inosine on the 3’ end of primer strongly destabilized PCR. Moreover, in case of multiple substitutions by inosine, modification in the first 5’ end of primer associated with contiguous degenerated residues clearly improved amplification. Further investigation will be done to confirm these effects.

Evaluation of DNA Fragmentation Methods for Implementation of cgMLST in Routine Analysis

Larissa Murr, Melanie Pavlovic, Ingrid Huber, Ulrich Busch, Patrick Guertler

Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764 Oberschleißheim, Germany

NGS currently represents the highest-resolution method for strain typing. In combination with a suitable analysis of the data, such as the comparison of the core genome multilocus sequence typing (cgMLST), NGS has the potential to differentiate the bacterial isolates of a species much more precisely than with the typing methods previously used in routine. Potential sources of infection can thus be identified more quickly. The use of NGS technology in routine analysis, as part of the official monitoring of food, requires an optimization of the corresponding workflow with regard to sequencing quality and time/cost ratio.

In addition to the decision whether to generate DNA libraries using PCR-free or PCR-dependent methods, the choice of the DNA fragmentation method is a crucial factor. Besides enzymatic methods, physical DNA fragmentation using ultrasonic is also possible. A key factor here is the generation of overlapping partial sequences (reads) that can be combined into coherent sequences after sequencing. The average number of reads per nucleotide position in the genome is called coverage and is a parameter for the quality and precision of the assembled sequence. The prerequisite for high coverage is the generation of strand breaks as randomly and uniformly as possible. Coverage can vary depending on the type of fragmentation method selected and the DNA sequence of the organism. Fragmentation, particularly in enzymatic processes, is not always completely random, but can occur with varying frequency in specific DNA sequences. Possibly, DNA libraries are created with an undesired wide size distribution with fragment sizes that deviate significantly from the desired length. Fragments that are too short or too long are removed during purification steps or size selection, which reduces the coverage of the affected DNA sequences or might lead to the fact that no sequence information at all is obtained for some DNA regions. Due to the short reading length during NGS sequencing, only parts of long DNA fragments are sequenced, which means that a lot of sequence information is lost.

In order to evaluate the susceptibility of different methods to the sequence-induced generation of irregular fragment sizes, their application to bacterial DNA with different GC contents (Campylobacter coli with ~30 % GC content and Salmonella enterica with ~50 % GC content) was tested in triplicate and evaluated in terms of time, cost and size distribution. Fragmentation was performed physically by ultrasonic on the one hand and enzymatically by tagmentation or two alternative enzymatic methods on the other hand. The generated fragments were separated by capillary electrophoresis and the size distributions of the individual isolates were compared before and after size selection. When enzymatic kits were used, the DNA could be overfragmented due to sequence-specific fragmentation, while physical fragmentation is easier to standardize due to less fragmentation bias.

FASTFISH-ID™: ThermaGenix’s Rapid DNA Authentication of Any Species of Commercial Fish Using Bio-Molecular Systems’ MIC sPCR Cycler.

Jesus Aquiles Sanchez1, Amanda M. Naaum2, Marine Cusa3, Ian Goodhead3, Sarah Helyar2, Albert Wijngaard4, Christopher Elliot2, Stefano Mariani3, Lawrence Wangh1

1Thermagenix, Inc,, Natick, Massachusetts, United States of America; 2Institute for Global Food Security, Queen’s University Belfast, Belfast, United Kingdom; 3School of Environment and Life Sciences, University of Salford, Salford, United Kingdom; 4Bio Molecular Systems, Upper Coomera, QLD, Australia

PROBLEM: Species mislabeling/substitution in commercial fish products threatens the economic welfare, product safety, and sustainability of the seafood industry. The sequence of the mtCO1 DNA barcode region serves as a universal molecular tag for species identification. COI DNA barcoding is not suitable for routine species monitoring along fish supply chains due to slow and costly off-site sequencing. SOLUTION: ThermaGenix’sFASTFISH-ID™ is a revolutionary low-cost DNA test that empowers anyone anywhere along the supply chain to rapidly authenticate ANY species of commercial fish without sequencing or having to do separate tests for each species. FASTFISH-ID™ provides answers in just two hours or less using a simple “sample-in, answer-out” test implemented in the MIC qPCR Cycler, a highly accurate, portable and affordable instrument from Bio Molecular Systems. METHOD: FASTFISH-ID™ converts species-specific COI DNA barcode sequences into highly-reliable fluorescent signatures that are then compared to a cloud-based reference library for immediate answers. Most importantly, FASTFISH-ID™ integrates seamlessly into existing regulatory DNA barcoding protocols: If needed, mislabeled and unknown samples rapidly identified with FASTFISH-ID™ can be directly sequenced for regulatory compliance or for reference library addition, respectively. Sample preparation is also fast: samples are just disrupted in a proprietary reagent and then diluted into the test for immediate identification. RESULTS: FASTFISH-ID™ was validated by two independent laboratories in England and Northern Ireland using a blinded panel of 18 commercial fish species comprised of 1-8 biological replicates (three technical replicates each) with 96%-100% success. FASTFISH-ID™ was also successfully tested on a research vessel in the North Atlantic. SIGNIFICANCE: FASTFISH-ID™’s turnkey “sample-in, answer-out” operation, portability, and cloud-based analysis software makes authentication of any fish species easier, faster, and cost-effective. Rapid and convenient FASTFISH-ID™ species authentication will protect the seafood industry against the vulnerabilities of species substitution/mislabeling. The same technology can be used for analysis of virtually any group of animals, plants, or microbes on Earth by designing the appropriate PCR primers and probe sets covering numerous genera and species.

Is The High-Throughput human OpenArray® System Useful For Profiling miRNAs In Melanoma Regression In A Swine Model?

Claudia Bevilacqua1, Fany Blanc1, Marie-Noelle Rossignol1, Guillaume Piton2, Sylvain Marthey1, Silvia Vincent-Naulleau2

1INRA, France; 2CEA, France

Cutaneous Melanoma (CM) is the most aggressive cancer of the skin due to its high metastatic potential. Despite recent encouraging results from target therapies, treatment of invasive melanoma is still challenging and new data on melanoma biology are required. MicroRNA (miRNA) are post-transcriptional regulators of gene expression. Altered miRNA expression profiles and the identification of their targets in cancer imply the high potential of miRNA as diagnostic and prognostic markers for development of new therapeutic strategies. The Melanoma-bearing Libechov Minipig (MeLiM) model is a relevant animal model of CM since melanocytic lesions are similar to human counterparts. MeLiM pigs are born with CM that progress to spontaneous regression leading to a high rate of survival even in metastasis-bearing animals. In this study, we investigated whether miRNA could be involved in this fascinating process.

Even if the release of the pig genome sequence in 2012 was a major step for using pig models for biomedical research, some data are always limited in swine. For example, in miRbase 21, only 382 sequences from Sus scrofa miRNA precursors are listed against 1881 for Homo sapiens. For this screening, we tested the use of TaqMan® OpenArray® human MicroRNA Panels (Pool A and B, ThermoFisher) charged with 750 human miRNA to compare quickly and easily, miRNA profiles between progressive and regressive MeLiM tumors. With 60% of positive amplification, results show that this approach could be an alternative of miRNA sequencing for miRNA screening in some species where “large” dedicated tools are missing.

New Advances in Two-Tailed RT-qPCR

Eva Rohlova1,2, Mansi Maheta1,2, Peter Androvic1,3, Lukas Valihrach1, Mikael Kubista1,4

1Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic; 2Faculty of Science, Charles University, Prague, Czech Republic; 3Faculty of Science, Palacký University, Olomouc, Czech Republic; 4TATAA Biocenter, Gothenburg, Sweden

MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as clinical biomarkers. In 2017, we presented a highly specific, sensitive and cost-effective method to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to termini of the targeted miRNA, connected by a hairpin structure. Here, we summarize the applications and advances in Two-tailed RT-qPCR in last two years. We used the two-tailed technology to create a panel for quality control (QC), monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The application of the QC panel is demonstrated on the optimization of RNA isolation procedure from biofluids. We also developed a protocol for simultaneous reverse transcription of mRNA and miRNA molecules in a single tube. In combination with direct lysis of cells in plates the protocol represents very time and cost-effective approach for quantification of miRNA and mRNA expression in in-vitro experiments.

New Versatile Enzymes for High Performance Multiplex RNA Detection and Analysis

Ryan Heller, Suhman Chung, Kyle Dumas, Kasia Crissy, Patrick Barchard, David Schuster, Thomas Schoenfeld

QIAGEN Beverly, United States of America

Reverse transcription PCR (RT-PCR) and variations like quantitative RT-PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR) are indispensable tools that are widely used for monitoring expression levels of disease-specific mRNA biomarkers, profiling noncoding RNA, and detecting pathogens, especially RNA viruses, at high specificities and sensitivities from biological and environmental samples. Despite their importance, no completely satisfactory RT is currently available. Most one step RT-PCR kits contain a mixture of two enzymes: a retroviral RT, usually a M-MLV derivative, and a thermostable DNA polymerase, usually derived from Taq polymerase. This reliance on two enzymes limits the temperature of cDNA synthesis and necessitates compromises in reaction conditions between those favorable for each component, which can hamper detection of certain RNA targets. A molecular screening and directed evolution program has provided single-enzyme hot-start RT-PCR solutions based on a new thermostable RT that is active at up to 85°C and stable up to 95°C. Higher temperature detection improves specificity and sensitivity by allowing direct detection of RNA targets at near-single-copy sensitivity. This is especially notable when detecting rare or highly structured targets. This new chemistry is compatible with singleplex and multiplex formats using most dye and probe-based detection chemistries. Detection facilitated by this enzyme is equally effective on RNA or DNA targets and can allow simultaneous detection of both without modification of the detection protocol. Because cDNA synthesis occurs rapidly during the initial stage of PCR, the elimination of a distinct RT step and inherently faster kinetics reduces detection times to less than 40 minutes. The inherent stability of the enzyme provides simplified formulation and improved liquid and dry storage. These improvements promise to simplify, accelerate, and improve the reliability and flexibility of detection and analysis of mRNA, noncoding RNA, and viral targets.

Polymerase Strand Displacement Reaction with SD DNA Polymerase – New Alternative to PCR

Anke Fenn, Ferdinand Holzinger, Konstantin Ignatov, Vladimir Kramarov, Andreas Kirsten, Sergey Kovalenko

Bioron, Germany

Polymerase Chain Displacement Reaction (PCDR) was described in 2013 by Harris et al (Biotechniques. 2013, 54(2), 93). It is a new technique combining thermocycling (like PCR) and isothermal (like SDA or LAMP) approaches to DNA amplification. For PCDR performing, two or more pairs of nested primers are used with a thermostable DNA polymerase that has strong strand displacement activity. When extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by strand displacement activity.

In contrast to PCR, new technique allows to synthesize several new DNA copies from the single template DNA molecule in each amplification cycle. Thus, PCDR is much faster and more effective than PCR. The assays based on this reaction are more sensitive. Besides, due to the use of several nested pairs of primers the specificity of the reaction is increased.

Till recently the main disadvantage of PCDR was the lack of commercially available thermostable DNA polymerase which is suitable for PCR and possesses strong strand-displacement activity. SD DNA polymerase (Bioron GmbH Patent US 9,896,671) allows to fill-in this gap and PCDR started to be a reality.

SD polymerase has been shown to direct effectively PCDR in several diagnostics application (Dengue virus detection, plant pathogen HLB detection). Moreover, recently the PCDR on the basis of SD polymerase has been developed further to so-called tailed tandem repeat PCDR (TTR-PCDR) which looks quite universal and can be applied for the fast and convenient detection of pathogens (J. Wang, et al. (2018) Anal. Methods (35), DOI:10.1039/C8AY01625A).

Thus, PCDR with SD polymerase is a promising method which outperforms PCR in speed and efficiency of amplification and can be used in laboratory practice for research and diagnostics purposes.

QPCR, DPCR and NGS for The Detection of EGFR Mutations In Lung Cancer Patients

Francesca Salvianti, Gemma Sonnati, Filomena Costanza, Pamela Pinzani

University of Florence, Italy

Lung cancer is a leading cause of cancer death worldwide. Patients with EGFR oncogene driving mutations benefit from treatment with tyrosine kinase inhibitors but eventually progress developing resistance to therapy. The most common mechanism of resistance is the secondary acquired mutation EGFR p.T790M. As a consequence, it is important to assesses EGFR mutational status at the time of diagnosis and during treatment. Molecular testing guidelines for selection of lung cancer patients for tyrosine kinase inhibitors indicate cell-free DNA (cfDNA) as a surrogate for the determination of EGFR status in patients with limited and/or insufficient tissue.

We performed a pilot study to compare different methods for the detection of EGFR mutations in cfDNA from a small cohort of lung cancer patients. In particular, we evaluated the performances of three approaches based on qPCR, dPCR and targeted NGS.

Results were mostly concordant, but some discrepancies were found for qPCR and dPCR. NGS in some cases allowed a more sensitive detection of variants, especially when a tag sequencing approach was adopted. The major advantage of NGS is represented by the possibility of a simultaneous detection of multiple targets. The pre-analytical phase, in particular cfDNA extraction methods, could also influence the results.

From our preliminary data emerges that the combined use of multiple methods allows a more comprehensive and reliable assessment of the presence of mutations in cfDNA.

SMARTer PicoPLEX Gold: A New Generation of Single Cell NGS Library with High Reproducibility, and Greatly Improved Coverage and Fidelity for Precision Medicine

Matthieu Pesant1, Fang Sun2, Datta Mellacheruvu2, Bayu Sisay2, John Langmore2, Graeme McLean2, Andrew Farmer2, Emmanuel Kamberov2

1Takara Bio Europe, Saint-Germain-en-Laye, France; 2Takara Bio USA, Inc., Mountain View, CA 94043, USA

Accurate, reproducible detection of mutations and copy number variations (CNV) from small amounts of DNA, including single cells and fixed tissue, is key for genetic analysis of clinical samples to assist in identifying the best treatment regimen and molecular diagnoses of diseases such as cancer. Increasing demand for genetic analysis from limited samples, including single cells, has created an increasingly unmet need for technologies that allow for economical and accurate analysis of those samples. A primary application for single-cell analysis is Preimplantation Genetic Testing (PGT). PGT has grown dramatically in the last ten years, enabled by improvements in the array and sequencing platforms, as well as by the patented SMARTer® PicoPLEX® WGA (PicoPLEX) quasi-random priming technology―the international gold standard for whole genome amplification (WGA) for subsequent detection of CNV in fixed or unfixed single cells. Initially, PicoPLEX chemistry was optimized to allow for reproducible detection of aneuploidies and CNVs in embryo biopsies. The original versions of the technology are not optimized for other applications such as genetic analysis in cancer screening, diagnosis of disease, or therapeutic drug monitoring due to the frequency of falsepositive mutation rates. To address the need for accurate detection of single nucleotide variants (SNVs), we enhanced the PicoPLEX chemistry using optimized enzymes, primers, and protocols that improve sequencing coverage, uniformity, and accuracy while increasing the resolution for CNV detection and retaining reproducibility. This enhanced chemistry named SMARTer PicoPLEX Gold Single Cell DNA-Seq Kit (PicoPLEX Gold) is a single-cell library-prep kit with a simple, four-step protocol to convert fixed or unfixed single cells into NGS libraries in under three hours with minimum hands-on-time. Libraries prepared from single GM12878 cells using the PicoPLEX Gold kit were sequenced on an Illumina® NextSeq® platform to a depth of ~35 million read pairs (2 x 150 cycles), generating >50% genome coverage. This coverage represents a 2-fold improvement over the original PicoPLEX kit, along with a 4X reduction in duplication rates. The PicoPLEX Gold kit detected 3.5X more SNVs compared to Multiple Displacement Amplification (MDA) with the same number of reads. Our proprietary highfidelity polymerases used in the PicoPLEX Gold kit produced up to 50% lower allele dropin (false-positive) rates than MDA. The increased coverage and low bias of the PicoPLEX Gold kit translated to extremely low allele-dropout rates (ADO), ~5X lower than MDA. Therefore, a single PicoPLEX Gold kit library enables reliable, high-resolution CNV analysis with shallow sequencing, and an accurate and reproducible SNV and CNV analysis with deeper sequencing.

Sample quality control of Cell-free DNA

Bettina Strauch, Eva Graf, Elisa Viering

Agilent Technologies, Germany

Sequencing of cell-free DNA (cfDNA) is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on preanalytical sample treatment or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of an automated electrophoresis platform performing cfDNA quantification with region analysis. Moreover, the results include a score to qualify cfDNA samples according to their contamination level with high molecular weight material. This allows defining a threshold for objective sample qualification prior to library preparation. The analysis features are described with examples of typical sample patterns.

SiPOOL: Fast, Reliable Gene Silencing With Exceptional Target Specificity Using Optimally-Designed Complex siRNA Pools

Catherine Goh1, Andrew Walsh1, Michaela Beitzinger1, Jonas Bertram1, Stefan Hannus1, Gunter Meister2, Michael Hannus1

1siTOOLs Biotech GmbH; 2University of Regensburg

RNA interference (RNAi) is widely used as a gene silencing tool to determine gene function. This is due to its ease of application, broad cell type applicability, drug-like properties and quick time to results. However, it is widely established that synthetic RNAi mediators, short interfering RNAs (siRNAs), can produce wide-spread off-target effects. This gives rise to highly variable results, necessitating time-intensive and costly validation efforts with multiple siRNA reagents. False positive results from siRNA off-targeting may also incur significant costs if left undetected. siPOOLs are complex pools of 30 optimally-designed siRNAs against a single gene. The high complexity pooling reduces the concentration of individual siRNAs, diluting siRNA-specific off-target effects. In contrast, target gene knock-down efficiency is increased due to the greater transcript coverage offered by a siPOOL. As a result, loss-of-function phenotypes become more robust and reproducible. siPOOL rescue constructs were also demonstrated to work efficiently to restore gene function. siPOOLs are ideal tools to ascertain gene function quickly and reliable and can be used in high-throughput RNAi-based screens to identify novel targets across various cell-based systems.

Small Circular RNAs With Antisense Function

Christina Pfafenrot, Albrecht Bindereif

Justus Liebig University of Giessen, Germany

Circular RNAs (circRNAs) are a relatively new class of non-coding RNAs comprised of hundreds of members, which are generated from pre-mRNAs by alternative splicing and are present in all eukaryotes investigated so far. Although a defined function of circRNAs is still under debate, some studies revealed a microRNA sponge function of naturally occurring circRNAs like ciRS-7 and SRY. In addition, several other, hypothetical roles have been discussed, such as templates for translation into peptides or proteins, protein sponging, allostery, scaffold functions in RNA-protein complex assembly, or antisense activity.

Here we describe a method for inhibiting translation initiation by small circular antisense RNAs. Translation of mRNAs is one of the mechanisms through which protein levels can be selectively regulated. The eukaryotic initiation factor eIF4E is one of the key regulators, which recognize the cap and forms a trimeric complex (termed elF4F) which is responsible for the activation of mRNA during the translation. This mRNA activation and also the translation can be inhibited by blocking the elF4F complex by binding of small antisense circRNAs to the 5'-untranslated region of mRNA, thereby impeding ribosome attachment and subsequent scanning of the mRNA. As a proof of principle, we designed and generated small synthetic antisense circular RNAs by in vitro transcription and ligation. HeLa cells were co-transfected with synthetic antisense circRNAs targeting ß-globin-luciferase-reporter constructs. Luciferase reporter assays were performed to measure translation activity in transfected HeLa cells. We could reach a translation reduction up to 60 %. Our results with synthetic designer circRNAs establishes that circular RNAs can function as antisense vectors, suggesting that natural examples of antisense circRNAs may exist. Furthermore, this suggests a possible therapeutic use of small circRNAs to inhibit translation.

Streamlined Single-tube Solutions for High Quality DNA Library Preparation

Yi Jing, Marissa Bolduc, David Bays, Shuhong Li, Eleanor Kolossovski, Brian Komorous, Hongbo Liu, David Shuster

Quantabio, United States of America

Simple, rapid, and reliable solutions for library preparation are in great demand as the next-generation sequencing (NGS) technology has become widely adopted in research, clinical and many other fields. Leveraging our innovative chemistries, world-class enzyme purity and rigorously controlled production and ISO13485 quality system, we have developed several high-quality library preparation solutions for Illumina platforms to address the speed, performance, and throughput requirements of different DNA-seq applications. The sparQ DNA Frag & Library Prep Kit supports easily tunable enzymatic fragmentation and end polishing in a single step, followed by high efficiency adapter ligation in the same tube without intervening purification steps. The simplified workflow combined with the novel chemistry lead to shorter hands-on time (30 minutes), higher library yield (up to 2 folds), better sensitivity, lower duplication rate, and greater coverage uniformity across a broad range of GC-content. For sample types not requiring initial DNA fragmentation, the sparQ DNA Library Prep Kit provides a fast, single-tube library preparation solution that ensures great sensitivity (down to 250 pg) and efficiency, while maximizing library yields. A high efficiency, low bias and high-fidelity PCR master mix was also developed to address applications that require accurate library amplification.

TATAA Biocenter - Your Full Range Commissioned Service Provider. Get Better Results Faster

Hanna Zoric, Jens Björkman, Mikael Kubista

TATAA Biocenter, Sweden

TATAA Biocenter offers full range of commissioned services and research projects, custom designed for your needs within the area of nucleic acid analysis (qPCR, dPCR, NGS, isolation/extraction, data interpretation) and more. We are a biotechnology company working at the forefront of molecular biology research delivering highest quality data using the most appropriate platforms based on more than two decades of experience within the field, including proprietary technologies such as Two-Tailed RT-qPCR[1] and SiMSen-Seq[2]. At TATAA, we also provide active tailored support throughout the whole lifetime of your project with special focus on quality control[3],[4],[5]. The work is performed in compliance with the International Standard ISO/IEC 17025:2005.

TATAA participates in the development of standardized procedures for molecular analyses within the EU initiative SPIDIA ( and the IMI initiative CANCER-ID (, of which the forthcoming CEN and ISO guidelines are based.

Example of services:

  • Gene expression profiling (RNA-seq, high throughput qPCR)
  • qPCR/dPCR assay design and validation
  • Rare event detection (dPCR, SiMSen-Seq)
  • miRNA profiling (EdgeSeq, Two-tailed RT-qPCR)
  • cfDNA analysis (dPCR, qPCR, NGS)
  • Copy number variation (dPCR)
  • Custom genotyping services (rhAmp and TaqMan chemistry)
  • High throughput Protein profiling (PEA chemistry, Olink Proteomics)
  • Single cell analysis (CellCelector, 10X)

Our customers find TATAA Biocenter services cost and time effective. Full projects can be outsourced as well as optimizations of workflows and protocols, in which case we train customer’s staff and transfer the methods and technologies to our client for use in their environment.

[1]Androvic et al. “Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification” Nucleic acids research vol. 45,15 (2017): e144.

[2]Ståhlberg et al. “Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing”Nucleic acids research vol. 44,11 (2016): e105.

[3]Bustin et al. “The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments”
Clinical Chemistry 55,4 (2009).

[4]Forootan et al. “Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR)” Biomolecular Detection and Quantification 12 (2017) 1-6

[5]Björkman et al. “Differential amplicons (ΔAmp)—a new molecular method to assess RNA integrity” Biomolecular Detection and Quantification, Volume 6, January 2016, Pages 4–12

The Spectrum Compact CE System: Fragment Analysis with a Personal Capillary Electrophoresis Device

Christopher D'Jamoos1, Ayaka Okuno2, Doug Storts1, Robert Mclaren1, Cynthia Sprecher1, Michiru Fujiocha2, Jin Matsumura2, Isao Haraura2, Asami Terakado2, Noriuki Sumida2

1Promega Corporation, Madison, WI 53711, USA; 2Hitachi High-Technologies Corporation, 882 Ichige, Hitachinaka-shi, Ibaraki-Ken, 312-8504 Japan

The Spectrum Compact CE System, a collaborative development between Promega Corporation and Hitachi High-Technologies Corporation, allows laboratories of all sizes the freedom to carry out single nucleotide polymorphism, PCR sizing and microsatellite analysis, de novo sequencing, NGS validation, and mutation detection. This four-capillary benchtop capillary electrophoresis instrument runs 1-32 samples and features single base resolution and 6-dye detection along with an integrated touch-screen for instrument operation. Pre-filled reagent cartridges, including a choice of separation polymers, streamline instrument setup and support both fragment analysis and sequencing workflows. This instrument is capable of detecting up to 6-colors with analysis using either GeneMapper® ID-X or GeneMarker® HID Software for Spectrum CE System. It is backwards compatible for all commercially available existing STR systems with performance comparable to current capillary electrophoresis instruments (e.g., sensitivity and mixture samples). Total electrophoresis time for fragment analysis is approximately 40-45 minutes. For the sequencing application, Promega has developed the Prototype ProDye™ Terminator Sequencing System. This is a dideoxy dye-terminator based sequencing chemistry that can generate readable sequences using a variety of templates. In combination with the Spectrum Compact CE System, read lengths of ≥ 600 bases can be obtained with run times of ≤ 32 minutes and ≥ 700 bases with a run time of ≤ 60 minutes.

The Synthetic DNA Fragment Use in the qPCR Diagnostics

Martina Grochová1,2, Karel Bílek1, Kateřina Rosenbergová1

1National Institute for NBC Protection (SÚJCHBO, v.v.i.), Czech Republic; 2Faculty of Sciences, University of South Bohemia in České Budějovice, Czech Republic

The aim of this presentation is to introduce the approach using a synthetic control sample in the qPCR-based detection strategies and point out the main advantages connected to its use. The term synthetic control is understood as a commercially synthesized double-stranded DNA of different length tailored to your target sequence or gene of interest.

Although the original intention is to offer ready-to-clone DNA fragments, this strategy can be especially useful considering the samples of biological species difficult to access. It could be the case of rare species or highly pathogenic viruses e.g. Ebola virus, SARS virus etc. The acquisition of such samples could be even impossible, or at least time-consuming concerning the complicated paperwork, and also very expensive when considering distant parts of the world. Additional reasons comprise the biosafety and biosecurity issues which are connected with the particular workplace, as well as the national legal restrictions.

To compensate for the lack of available samples, the synthetic control represents a suitable way to validate the assay without a real sample of the target agent. Thanks to the fact that the control can be commercially synthesized within a few days, it is possible to order a new stock of your target very fast and easy. Moreover, the control is prepared highly concentrated ensuring a sufficient amount of working solution. The dilutions can be used as a concentration standard for further assay validation according to the MIQE guidelines. However, certain limitations exist comprising the length, complexity of the sequence, GC content etc.

To support the presented idea, the examples of particular assays will be mentioned, as well as the workflow involving the design of synthetic control, starting from the thorough in silico study based on available target sequences in the Genbank database, up to the order placement. Thus, it is demonstrated an efficient tool for the set up of a synthetic positive control in the detection of uncommon samples or simply as a useful concentration standard for the validation of qPCR assays.

This work was supported by the Ministry of Interior of the Czech Republic, no. VI20172019063.

ThermaGenix’s Universal PCR Additives for Suppressing DNA and RNA Amplification Errors.

Jesus Aquiles Sanchez, John Rice, Kenneth Pierce, Lawrence Wangh

Thermagenix, Inc,, Natick, Masaschusetts, United States of America

The Polymerase Chain Reaction (PCR) is a mainstay of molecular biology and genomics. Mispriming errors during setup and PCR compromise the specificity and sensitivity of DNA and RNA amplification and represent a major obstacle for rapid development of accurate PCR tests. ThermaStop™, ThermaGo™, and ThermaStop-RT™ are novel, easy-to-use universal additives that suppress mis-priming errors before, during, and after PCR for reactions starting with either DNA or RNA. Unlike commonly used antibody and chemical-based hot-start reagents, ThermaStop™, ThermaGo™, and ThermaStop-RT™ are not irreversibly inactivated by heat. Instead, these additives renature upon cooling and then bind to all Type A and Type B DNA polymerases (or reverse transcriptases in the case of ThermaStop-RT™) to enhance their specificity in a temperature-dependent manner; see for a full list of enzyme tested. ThermaStop™ is a hot-start reagent that outperforms hot-start Taq DNA polymerases. ThermaStop™ makes DNA polymerases more specific in their use of correctly matched primers and suppresses primer dimer formation in no template controls and in amplification reactions with target DNA. ThermaStop™ also reduces the time required to build multiplexed reactions, makes multiplexed reactions more accurate and informative, and acts synergistically with standard antibody-based hot-start for both Type A and Type B DNA polymerases. Complete master mixes containing all reaction components, target DNA, hot-start antibody, and ThermaStop™ remain inactivated for days or months, depending on the temperature at which they are stored prior to being heated the first time. ThermaStop™“cold-stop” activityalso preserves the integrity of amplification products after PCR and enhances the quality of all downstream applications, including NGS, deep sequencing, and Gibson assembly. ThermaGo™ acts synergistically with ThermaStop™ to improve amplification specificity. ThermaStop-RT™ is a first-in-class hot-start reagent that controls the low temperature activity of MMLV and AMV derived reverse transcriptases used for cDNA synthesis in One-Step and Two-Step RT-PCR. ThermaStop-RT™ allows for use of less reverse transcriptase, enhances the accuracy of highly multiplexed RT-PCR assays, and permits quantitatively accurate measurements of very low levels of RNA. ThermaStop™, ThermaGo™, and ThermaStop-RT™ deliver High Precision PCR and RT-PCR by promoting more efficient use of primers, higher levels of intended products, and increased sensitivity and quantitative accuracy for both monoplex and multiplexed PCR and RT-PCR assays. ThermaStop™, ThermaGo™, and ThermaStop-RT™ are commercially available from ThermaGenix’s, Inc., Natick, MA.

Use of Single Control DNA to Internally Evaluate Performance of qPCR and dPCR Instruments

Dejan Stebih, Katja Stare, Marjana Camloh, Mojca Milavec

National Institute of Biology, Slovenia

Calibrated and properly maintained equipment is the prerequisite for reliable measurements. Maintenance and operational qualification of real-time quantitative PCR (qPCR) and digital PCR (dPCR) equipment can become very costly, particularly when (1) there are several instruments in a laboratory, (2) when checking needs to be done more than once per year or (3) when instruments are rarely used. However, complete operational qualification is not always necessary, e.g. when instruments are moved or minor repairs are done.

Maintenance and operational qualification procedures and their time intervals for instruments have to be defined by each laboratory according to its needs, manufacturer recommendations, frequency of use and knowledge about the instruments. Here we present an approach that enables regular in‑house checks of instruments by trained laboratory personnel.

To enable a successful implementation of this approach we have prepared a control DNA that enables us to check the performance of different qPCR (LightCycler® 480 System, Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System, ViiA 7™ Real-Time PCR System and 7900HT Fast Real-Time PCR System) and dPCR platforms (Bio-Rad QX100™ and QX200™ Droplet Digital™ PCR Systems, including AUTO DG and Fluidigm Biomark™ HD), after minor repairs, as an intermediate check up between operational quantifications or as a yearly check. Following DNA extraction from the reference material (MON87769, AOCS 0809-B), using CTAB method, two European Union Reference Laboratory validated assays targeting (1) soybean reference gene – lectin, and (2) genetically modified soybean line - MON87769, were used for evaluation of control DNA. After operational qualification of QX100™ Droplet Digital™ PCR System, copy number per 1 µL of DNA solution for both amplicons, using singleplex and duplex reactions has been assessed and criteria for acceptance were determined.

To check the performance of dPCR platforms, control DNA is analysed with both singleplex and duplex assays targeting lectin and MON87769. To check the performance of qPCR platforms, standard curve is prepared from control DNA and analysed using singleplex assays only. To observe possible differences between different parts of the cycler block, reactions are spread across a 384-well PCR plate

The presented approach serves as a complement to the external service, enabling us to maintain instruments performance between external qualifications and reduces the cost of checks between external testing.

MOL-PCR and xMAP Technology: Fast Multiplex Method with High Sensitivity

Jirina Markova, Petr Kralik

Veterinary Research Institute Brno, Czech Republic

The world of diagnostics goes still forward and puts demands on laboratories especially in diagnostic of important infective agents. Methods for multiplex detection are mainly designed for relatively fast and cost-effective examination of wide spectrum of different analytes to save money and time and give a complex results. xMAP (x=analyte, MAP= Multi Analyte Profiling) technology provides qualitative analysis of various proteins or nucleic acids simultaneously in a single reaction and enables the direct detection, identification and typing of more than 50 different targets from one biological sample.

This technology is based on multiplex oligonucleotide ligation – polymerase chain reaction (MOL-PCR) with an adaptation to xMAP detection system using different sets of microsphere in a liquid suspension. These are dyed of red and infrared fluorophores. The concentration of internal dyes gives to each set its unique spectral address. During MOL-PCR, a pair of probes (MOLigo1 and MOLigo2) designed specific for target sequence binds to a searched part of analyte and the process of their ligation creates templates for amplification in PCR where the universal primer pair (one has a fluorescent label) is used. The specific part of one MOLigo probe contains the unique TAG sequence – oligonucleotide sequence complementary with an anti-TAG sequence covalently coupled on the microsphere surface. Thanks to this xTAG technology, amplified denaturized products hybridize to corresponding microspheres set. Use of different TAG/anti-TAG sequences enables multiplex and qualitative sample analysis in MAGPIX® instrument (Luminex Corporation) where the medium fluorescence intensity (MFI) is measured.

During routine analysis, xMAP technology thus brings an opportunity to very quick identification of diverse targets (from gene expression monitoring to detection of nucleic acids, proteins, saccharides and another macromolecules) using small amount of examined material.

This work was supported by Security Research of Ministry of the Interior of the Czech Republic VI20152020044.

Exteme One-Step RT-qPCR: Potential for point-of-care viral detection.

Jessica A. Houskeeper, Lauryn Narramore, Carl T. Wittwer

Univeristy of Utah, United States of America

Introduction: Reverse transcription (RT) is often coupled with qPCR for simplified detection of RNA. Despite the ease of One-Step RT-qPCR reactions, some pathogens are still assessed using a Two-Step method. Most Two-Step methods require over an hour for pathogen identification. Even the best commercially available One-Step RT-qPCR kits require a minimum of 20 minutes for detection. Due to the essential nature of fast and accurate detection of viral infections, we have developed a One-Step RT-qPCR assays that can be performed in under 1 minute.

Methods: A prototype instrument was used to temperature cycle 5 µL samples in 0.4-1.0 seconds at annealing, extension, and denaturation temperatures. To transition rapidly between temperatures, reactions required increased polymerase, primer, and Mg++concentrations. Gene specific primers were designed with melting temperatures between 60-75˚C. Detection sensitivity and efficiency of 5 different viral targets were analyzed by real-time PCR. Sample specificity was demonstrated by high-resolution melting.

Results: Viral targets acquired from the American Type Culture Collection (ATCC) were amplified as 50- to 100-bp targets in a minute or less. Without the use of hot start methods, the Extreme RT-qPCR assay produced no-template controls with realtively early amplification. This was attributed to the increased reaction component concentrations resulting in enhanced non-templated additions. However, with the implementation of hot start techniques, non-templated additions were significantly reduced prior to thermocycling as confirmed by real-time PCR and high-resolution melting.

Conclusions: Use of Extreme diagnostic technology has led to the design of a One-Step RT-qPCR assay that can be performed in under a minute with competitive sensitivity to alternative RT-qPCR methods. Extreme RT-qPCR illustrates the ability of point-of-care diagnostic testing for infectious disease when prompt results may be critical.

QC Measurements for Predicting Downstream NGS Success with FFPE and Circulating Cell-Free DNA Plasma Samples

Christopher D'Jamoos, Doug Wieczorek, Spencer Hermanson, Curtis Knox, Jennifer Mook, Eric Vincent, Doug Horejsh, Trista Schagat, Doug Storts

Promega Corporation, 2800 Woods Hollow Rd. Madison, WI

Introduction: Quantity and quality of DNA from formalin fixed, paraffin embedded(FFPE) tumor tissue samples is highly variable, with degradation and crosslinking due to the fixation process leading to issues with amplification and difficulty in NGS analysis. An alternative to FFPE is circulating cell-free DNA (ccfDNA) from plasma or other biological fluids. Compared to gDNA, ccfDNA yields are typically low, with tumor cell present at significantly lower frequencies. Due to the inherent variability of FFPE and ccfDNA, knowing the quantity of DNA is not in itself reliably predictive of downstream NGS success. In this poster, we describe novel methods for predicting sequencing result quality utilizing a multiplexed qPCR assay. Methods: DNA was purified from four matching tumor and normal FFPE tissue types as well as ccfDNA from plasma samples using multiple methods. DNA quantity was measured via single-target qPCR and used for downstream NGS library construction with a 56 gene oncology panel and subsequent data analysis. Discrepancies between quantity of DNA input into library preparation and expected library yield and sequencing coverage uniformity were noted. To investigate if downstream library yield and sequencing quality could be better predicted, a multiplexed qPCR assay was designed that included three different amplicon sizes (75, 150, and 300bp). The quantitative differences between the increasingly larger amplicon sizes were calculated as a ratio to determine the level of degradation of the DNA from FFPE samples. ccfDNA fragments cluster around 170bp; thus, the 150bp target closely estimates ccfDNA concentration. Since gDNA is expected to be much larger in size, the ratio of 150bp to 300bp targets can help predict the ratio of ccfDNA to gDNA. FFPE samples with high 75/300bp ratios are indicative of highly degraded samples, and ccfDNA samples with low 150/300bp ratios are indicative of gDNA contamination. Results: Retroactive testing with the multiplexed qPCR assay showed a strong correlation of degraded DNA to low library yield and low coverage uniformity. Samples with equal concentration and library input performed much better when degradation ratios were low. Conclusions: Data derived from a multi-size target qPCR assay can be very effective in predicting downstream NGS success. Using such a QC method can drive researchers to triage samples and make informed decisions about what downstream library method to use. Concentrating on less complex panels or ddPCR vs. highly multiplexed panels or whole exome sequencing for degraded samples can ensure getting the most useful information out of an individual sample, thus saving time, cost, and loss of information about precious samples.

Combination of Hormone and Transcriptome Analyses in the Study of Cold and Freezing Stress in Grass Lolium perenne

Sylva Prerostova1, Barbara Kramna1,2, Jan Simura3, Alena Gaudinova1, Vojtech Knirsch1, Ondrej Novak3, Radomira Vankova1

1Institute of Experimental Botany, Czech Academy od Sciences, Czech Republic; 2Faculty of Science, Charles University, Czech Republic; 3Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University and Czech Academy of Sciences, Czech Republic

Plants in temperate zone have to adapt to the changing temperature during year seasons. Cold and freezing temperatures cause misbalance in metabolism (stress). The plant responses to environmental changes are driven by phytohormones. In our study, we have used frost-sensitive and frost-tolerant clones of ryegrass Lolium perenne in order to describe changes in their metabolism associated with high stress tolerance. Plants were exposed to frost treatment (-7°C for 4 d), either directly or after cold acclimation (3°C for 7 d). Samples of leaves, crowns (meristematic tissue) and roots were analysed by UHPLC–ESI–MS/MS to determine the content of 87 hormone metabolites and by RT-qPCR for transcription analysis. The transcription of selected stress-related and hormone-related genes was correlated with the content of phytohormones.

The resistant clone had lower basal level of cytokinins (CKs) as well as the lower transcription of CK biosynthetic genes (LpIPT5 and LpIPT8) in roots. Contrarily, the transcription of stress-related genes LpIRI3 and LpIRI4, and abscisic acid (ABA) biosynthetic gene LpNCED1 was higher than in the sensitive clone. Cold acclimation was associated with elevation of CK levels and transcription of LpIPT genes, especially in crowns. Also, ABA and salicylic acid (SA) levels were up-regulated in all organs. The strong effect of the clone frost resistance was observed in the case of auxin. Subsequent frost led to increase of CKs in leaves, but decrease in crowns. The content of ABA and SA decreased, substituted in roots and crowns by the higher content of jasmonic acid (JA). Direct frost had a negative impact on highly-active CKs and it stimulated accumulation of low-active forms, as well as ABA and SA. The level of JA was in this case suppressed in roots and crowns but highly up-regulated in leaves. The transcription pattern of hormone-related genes generally correlated with the levels of phytohormones, few exceptions will be described. The transcription of stress-related genes differed significantly between tolerant and resistant clones, mainly in the case of LpCBF3, LpIRI3 and LpIRI4. In conclusion, our results show that the content of phytohormones differs dramatically in the situation of direct frost or freezing after acclimation. The regulation of the responses is in large part caused by different regulation of hormone metabolism and signalling.

Acknowledgement: The work was supported by the Czech Science Foundation, project no. 17-06613S.

Differentiation Potential of NG2-glia Following Different Types of Brain Injuries

Lukas Valihrach1, Denisa Kirdajova2, Denisa Kolenicova2, Daniela Krocianova2, Jan Kriska2, Peter Androvic1,3, Daniel Zucha1,4, Eva Rohlova1,4, Mansi Maheta1,4, Miroslava Anderova2, Mikael Kubista1,5

1Institute of Biotechnology, Czech Republic; 2Institute of Experimental Medicine, Czech Republic; 3Faculty of Science, Palacký University, Czech Republic; 4Faculty of Science, Charles University, Czech Republic; 5TATAA Biocenter, Sweden

NG2-glia, a fourth major glial cell population, are present in the adult central nervous system and display distinct morphology, antigens and functions from other mature glial cell types. Recently, many studies have shown that these cells are multipotent in vitro and they also display wide differentiation potential under pathological conditions in vivo. The aim of this study was to identify differentiation capacity and rate of proliferation of NG2-glia after different types of brain disorders, such as focal cerebral ischemia (FCI), stab wound (SW) and demyelination (DEMY). We used transgenic Cspg4-cre/CAG-tdTomato mice, which after administration of tamoxifen express red fluorescent protein (tdTomato) in NG2-glia and cells derived therefrom. Differentiation and proliferation potential of tdTomato positive (tdTomato+) cells in sham-operated mice and those after injury were determined using single cell RT-qPCR and immunohistochemistry. We identified two main populations (NG2-glia, oligodendrocytes) which each had 4 subpopulations. Expression profiles showed that one subpopulation (astrocytic like NG2-glia) were present only after FCI not after DEMY or SW what confirmed our data of immunohistochemistry. Moreover, this subpopulation had a high percentage of cells expressing Smoothened, a key factor of Shh signaling. Taken together, we have described the ability of NG2 cells to acquire multipotent phenotype after different types of injury and that the phenotype fluctuates depending on the pathological condition.

Metrological Support for Molecular Diagnostics - A Case Study of HCMV and Its Resistance to Ganciclovir

Alexandra Bogožalec Košir1, Tašja Cvelbar2, Mojca Milavec1

1National Institute of Biology, Department of Biotechnology and Systems Biology, Slovenia; 2Biotechnical Faculty, Department of Biology, University of Ljubljana, Slovenia

Methods for fast, accurate and sensitive detection, identification and quantification of pathogens are of paramount importance for reliable and effective disease control. While nucleic-acid amplification‑based methods offer great potential in clinical diagnostics, reproducibility and comparability of results are hampered due to the lack of reference methods and reference materials. One of the challenges of metrology, the science of measurement, is the establishment of support for reliable measurement of nucleic acid concentration.

To investigate the potential of application of metrology in the development of methods for molecular diagnostics, resistance of human cytomegalovirus (HCMV) to antiviral drug ganciclovir (GCV) was selected as a case study. HCMV or human herpesvirus 5 (HHV5) is a 230 kB double stranded DNA virus infecting monocytes, macrophages and dendritic cells. HCMV often leads to wide spread of viral replication and dissemination to multiple organs and can be life threatening to individuals with compromised or immature immune system. Antiviral drugs, predominantly GCV, are used to treat patients and prevent further dissemination of the virus. As the development of resistance can be life threatening especially for immunocompromised patients, a reliable method for detection and monitoring of infection and viral load is necessary.

The aim of our research was to develop and assess in terms of sensitivity, repeatability, linearity, dynamic range and robustness, nucleic-acid amplification‑based methods targeting three of the most common point mutations that contribute to GCV resistance in HCMV. In recent years digital PCR (dPCR), has become widely used in the field of nucleic acid analysis, including viral diagnostics. Although, dPCR is a prosperous methodology, the majority of diagnostic laboratories rely on real-time quantitative PCR (qPCR). Thus, the developed methods were assessed both on dPCR and qPCR platforms in order to determine the suitability of different platforms for molecular diagnostics. This research was financially supported by the AntiMicroResist project (the EMRP project, which is jointly funded by the EMRP participating countries within EURAMET and the European Union).

Detection of Bacteria and Virus Involved Urinary Tract Infections and Profiling Their Antibiotic Resistance Using TaqMan Real-time PCR Assays

Pius Brzoska, Xiaoqing You

Thermo Fisher Scientific, United States of America

Urinary tract infections (UTI) are commonly caused by the gram negative bacteria Escherichia coli, Klebsiella pneumoniae and the gram positive Staphylococcus epidermidis, and Enterococcus faecalis. Other less frequent bacteria causing UTIs are Morganella morganii, Acinetobacter baumanii, Citrobacter freundii and others. Some viral and fungal pathogens also have been associated with UTI.

We developed comprehensive panel of real time PCR assays to detect different bacteria, virus and fungi involved in UTI. The panel is based on the TaqMan® assay platform and was designed to allow the specific detection of these pathogens on the microfluidic OpenArray® platform. A bioinformatic assay design pipeline was developed to enable the design of specific and sensitive detection assays taking under consideration genome sequences of 80,000 bacterial species deposited at NCBI.

UTI infections are often resistant to antibiotic treatment. Carbapenem resistant and extended spectrum beta lactamase harboring bacteria are most commonly found in UTI. Using our assay design pipeline we also developed a panel of TaqMan® assays to detect genes conferring antibiotic resistance. We focused on beta lactamases and included detection assays for a wide variety of different members of this group, including the OXA, PER, NDM, CTX and other beta lactamases. Also assays for predictive identification of vancomycin resistance and quinolone resistance are included. The combined panel allows the identification of UTI associated microorganisms and their antibiotic resistance genes on the microfluidic OpenArray®system.

Immunomodulatory Properties of Adipose-Derived Stem Cells Treated with 5-Azacytydine and Resveratrol on Peripheral Blood Mononuclear Cells and Macrophages in Metabolic Syndrome Animals

Krzysztof Marycz1,2, Katarzyna Kornicka1, Agnieszka Smieszek1

1Wrocław University of Environmental and Life Sciences, Poland; 2Faculty of Veterinary Medicine, Equine Clinic-Equine Surgery, Justus-Liebig-University, 35392 Gießen, Germany

Endocrine disorders, including equine metabolic syndrome (EMS), are a serious issue in veterinary medicine and horse breeding. Furthermore, EMS was shown to affect the cytophysiological properties of adipose-derived stem cells, reducing their therapeutic potential. However, it was shown that those cells can be rejuvenated while using a combination of two chemicals: 5-azacytydine (AZA) and resveratrol (RES).In the present study, we decided to evaluate the immunomodulatory properties of AZA/RES-treated adipose-derived stem cells (ASC) isolated from EMS horses (ASCEMS). Thus, we co-cultured ASC with peripheral blood mononuclear cells (PBMC) and RAW264.7 macrophages. Most attention was placed on regulatory T lymphocytes (TREG), as well as the messenger RNA (mRNA) and protein levels of several cytokines(tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-10, and IL-1β). Moreover, we also investigated the expression of genes related to auto- and mitophagy in both PBMCs and ASCs. PBMCs were obtained from healthy and EMS-suffering individuals and were co-cultured with ASCs that were isolated from healthy and EMS horses cultured in control conditions and with AZA/RES. We discovered that cells treated with AZA/RES increase the TREG number while co-cultured with PBMCs. Moreover, the co-culture of PBMCs with AZA/RES-treated ASCEMS induced mitophagy in PBMCs. Furthermore, ASCEMS pre-treated with AZA/RES displayed anti-inflammatory properties, as decreased levels of TNF-α, nitric oxide (NO), and IL-6 were observed in those cells in comparison with their untreated counterparts in the co-culture with RAW264.7 macrophages. In summary, we demonstrated that ASCEMS treated with AZA/RES displayed increased anti-inflammatory properties, and was able to regulate and activate the TREG-related anti-inflammatory response.

Elucidation of the Role of Tenacibaculum spp. in Atypical Winter-ulcer in Sea-farmed Atlantic Salmon in Norway

Bjørn Spilsberg, Karin Lagesen, Hanne Katrine Nilsen, Anne Berit Olsen, Duncan Colquhoun

Norwegian Veterinary Institute, Norway

The disease ‘tenacibaculosis’ otherwise known as ‘atypical winter ulcer’ has become increasingly common in Norwegian Atlantic salmon farming within the last decade. This disease is primarily characterised by severe tissue -necrosis and -loss affecting the head and/or fins of infected fish. Losses related to tenacibaculosis are typically acute and may be extremely high. Incidences of up to 80% mortality are not unheard of and in severe cases affected stocks may be destroyed due to welfare concerns and to avoid possible transmission of infection following the precautionary principle.

The reasons for the recent emergence of tenacibaculosis as a common and severe disease in Norwegian salmon farming remain unknown. It is, however, well recognised that most diseases affecting farmed fish are a result of the interplay between environment, host species and pathogenic agent i.e. the presence of the ‘pathogen’ alone may not necessarily lead to manifestation of disease.

Tenacibaculum species play an important ecological role in recycling marine detritus and more than 25 species have been validly described, with many more remaining as yet unstudied. In an effort to identify a possible aetiological role of particular Tenacibaculum species/strains in development of ‘atypical winter-ulcer’ we sequenced 90 Tenacibaculum isolates collected from 15 geographically distinct disease outbreaks on a MiSeq. The primary aim was to generate a phylogeny over the strains including publically available Tenacibaculum sequences in NCBI.

Investigation Of Association Of Variant Rs17036314 Of PPARG Gene And Rs7923837 Of HHEX Gene In Diabetes Mellitus Type 2 Patients In Iran Population By Tetra-ARMS PCR And Sanger DNA Sequencing

Maryam Majzoobi

IAUPS, Iran, Islamic Republic of

In recent decades, Diabetes mellitus type 2 is a complex metabolic disease developed by interactions between various genetic factors and environmental factors in adult individuals of Iranian population of variable ethnicity. In this study, genetic investigation of PPARG and HHEX genes in association with diabetes mellitus type 2 disease is elaborated. Genetic study of the association of rs17036314 in PPARG and rs7923837 in HHEX genes with diabetes mellitus type 2 disease is performed by Tetra primer ARMS PCR technique, a frequently used, precise, low cost method for detection of mutations in single nucleotide polymorphisms in involved genes. Study of rs17036314 in PPARG gene (G>C) in 70 patients affected by diabetes mellitus type 2 and 70 normal subjects indicates 54.28% of patients with mutant allele and 45.71% with normal allele. Study of rs7923837 in HHEX gene (A>G) in 70 patients affected by diabetes mellitus type 2 and 70 normal subjects indicates 58.35% of patients with mutant allele and 13.6% with normal allele. Tetra primer ARMS PCR for genotype determination of rs17036314 in PPARG and rs7923837 in HHEX genes is benefited for selection of proper drug therapy, better prognosis of disease, risk assessment of diabetes mellitus type 2 and diagnosis of the disease.

Microvesicles Derived From Human Adipose-Derived Multipotent Stromal Cells Improves Retinal Functionality in Dogs With Retinal Degeneration.

Agnieszka Śmieszek1, Anna Pakuluk2,3, Natalia Kucharczyk2,3, Peter G.C. Bedford4, Krzysztof Marycz1,5

13Department of Experimental Biology, The Faculty of Biology and Animal Science, University of Environmental and Life Sciences Wroclaw 50-375, Poland; 2Przychodnia weterynaryjna Trzebnicka; 3Przychodnia weterynaryjna Viva; 4Professor Emeritus of Veterinary Ophthalmology, Royal Veterinary College, London, UK; and Ophthalmology Referrals, 25, Great North Road, Brookmans Park, Herts. AL9 6LB, UK; 5Faculty of Veterinary Medicine, Equine Clinic-Equine Surgery, Justus-Liebig-University, 35392 Gießen, Germany

The aim of the study was to determine the influence of microvesicles (MVs) derived from multipotent stromal cells isolated from human adipose tissue (hASCs) on retinal functionality in dogs with various types of retinal degeneration. The biological properties of hASC-MVs were determined using an in vitro model of retinal Muller-like cells (CaMLCs). The influence of hASC-MVs on cell viability and metabolism and brain-derived neurotrophic factor (BDNF) was tested. Biological activity of hASC-MVs was performed under normal and oxidative stress conditions. Preliminary clinical studies were performed on nine dogs with different types of retinal degeneration. The clinical studies included behavioural tests, fundoscopy and electroretinography before and after hASC-MVs intra­vitreal injection. The in vitro study showed that CaMLCs treated with hASC-MVs were characterised by improved viability and mitochondrial potential, both under normal and oxidative stress conditions. Additionally, hASC-MVs under oxidative stress conditions reduced the number of senescence-associated markers, correlating with the increased expression of BDNF. The preliminary clinical study showed that the intra­vitreal administration of hASC-MVs significantly improved the dogs’ general behaviour and tracking ability. Furthermore, fundoscopy demonstrated that the retinal blood vessels became less attenuated and electroretinography using HMsERG demonstrated an increase in a- and b-wave amplitude after treatment. These results shed promising light on the application of cell-free therapies in veterinary medicine for retinal degenerative disorders treatment.

Strigolactone Mediated Age - And Organ-Specific Gene Expression In Arabidopsis thaliana

Barbara Kramna1,2, Sylva Prerostova1, Eva Kobzova1, Radomira Vaňkova1

1Institute of Experimental Botany, Czech Acad. Sci.,; 2Charles University in Prague, Faculty of Science, Department of Experimental Biology of Plants

Inorganic phosphate (Pi), one of the most important macronutrients for plants, is often difficult to obtain due to its low solubility and a general lack in the soil. Nutrient stress conditions represent a serious threat to the environment and agriculture. Strigolactones (SL) are phytohormones, which orchestrate plant responses to the lack of Pi, including regulation of shoot branching and root architecture. The effect of synthetic SL analogue GR24 on gene expression was investigated in Arabidopsis thaliana during Pi starvation and in full Pi nutrition (100 µM). Plants were grown in hydroponics at the presence of 5 µM GR24 in a 7-day experiment. Genes of interest included 3 groups: SL-related; Pi transporters and the antioxidant system-related ones. The aim has been to compare transcriptomic responses in younger (3-week) and older (5-week) plants. The focus was also given to organ-specific (leaves and roots) expression patterns.

GR24 application at full Pi nutrition led in leaves to over-all up-regulation of strigolactone-related genes in older plants, when compared to the younger ones. In roots, more profound up-regulation of several biosynthetic genes (D27; MAX1) and transport ones (PDR1/PDR2) was observed after GR24 application also in older plants. Repressors of SL signal transduction SMXL2/6/8 were up-regulated as well, which might have been caused by positive-feedback regulation of strigolactone signalling pathway. The data suggest that the responses of older plants to exogenous strigolactone GR24 are stronger than those of younger ones. Antioxidant system-related genes were also more up-regulated after GR24 application in older plants, both in leaves and in roots. Phosphate transporters showed a different response in leaves, where PHO1 was down-regulated in older plants, while the opposite trend was found in roots. Also, both PHT transporters (PHT1.4; PHT1.7) were up-regulated in roots after GR24 application. These GR24 responses were strengthened at Pi deficiency.

Results suggested that older plants are not only more sensitive to exogenous GR24 application but that these transcriptomic changes are associated with up-regulation of antioxidant system-related genes. Moreover, we observed that transcriptomic response to GR24 treatment was organ-specific.

Acknowledgement: The study was supported by the Charles University, project GA UK No 1086217.

Role of Nitric Oxide During Embryonic Wound Healing

Pavel Abaffy, Ravindra Naraine, Silvie Tomankova, Radek Sindelka

Laboratory of Gene Expression, Institute of Biotechnology, Czech Academy of Science, Czech Republic


The long-term goal of our team is to determine the mechanism of embryonic wound healing and regeneration. In adults, wound healing is closely associated to the role of nitric oxide (NO), which is a known regulator of inflammation, angiogenesis and matrix metalloproteinases. However, at a certain stage of development embryos lack an immune system and vascular system but are still able to have complete healing of wounds. Herein we present the role of NO in embryonic wound healing.


We used early embryos of Xenopus leavis as our model to study wound healing. Utilizing RNA-Seq methodology, we determined changes of gene expression during this process and using functional assays the effects after inhibition of NO production.


Inhibition of the NO pathway during and after injury of X. laevis embryos led to defective wound healing. Only a small number of genes were found to be involved in the response to injury and were altered when NO was inhibited. Some of these genes (i.e.: aqp3, mmp8, lep) are known to be connected with defence response, which is very interesting given that the embryo is lacking an immune system.


We concluded that NO is important for the regulation of embryonic wound healing and the regulation of embryonic pseudo-inflammatory system.

Study Of An In Vitro System For Alzheimer’s Disease

Luo Wei2, Larry Wong1, Deanna Woo2, Matthew Hammond2, Laura Moriarty2, Elisabeth Jordan Dreskin2

1Bio-Rad Laboratories, Hercules, CA, USA; 2Bio-Rad Laboratories, Mississauga, ON, Canada

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases, which accounts for over 80% of dementia cases in people over 65 years old. The rapid increase in AD cases not only has a significant impact on the economy, but also imposes huge burdens on families and society due to the disease rendering patients dependent upon others for their health and safety. Many disease models have been established to study AD, including animal models, primary cultures, stem cells models, and cancer cell models (Carolindah et al. 2014). We sought to establish an AD model system in our lab, using human neuroblastoma cells (SH-SY5Y) which can be differentiated into mature neuron-like cells. In order to lidate this system, we used microscopy, RT-qPCR, and multiplex immunoassays to study the expression of key neural biomarkers, including AD markers. Additionally, we screened a long noncoding RNA panel to identify lncRNAs that are differentially expressed during the transition of these cells from neuroblastoma to neuronal cells. However, during the course of our study, we uncovered significant differences between the two SH-SY5Y cell lines we were working with. The two cell lines were sourced from two different suppliers, and we found they were actually very different to each other in terms of gene and protein expression and cytokine secretion.

The Expression of Prostaglandins during different Physiological stages in the Bovine Corpus Luteum

Bajram Berisha1,2, Dieter Schams2, Daniela Rodler3, Fred Sinowatz3, Michael Pfaffl2

1University of Prishtina, Kosovo; 2Animal Physiology & Immunology Weihenstephan, Technical University of Munich, Germany; 3Department of Veterinary Sciences, Ludwig Maximilian University of Munich, Germany

The ovarian cycle in ruminants is characterized by repeated patterns of specific cellular proliferation, differentiation and transformation that accompanies follicular development and corpus luteum (CL) formation and function. The objective of this study was to characterize some prostaglandin family members in bovine CL during estrous cycle and pregnancy. The CL tissue was assigned to the following stages of estrous cycle: 1-2, 3-4, 5-7, 8-12, 13-16, >18 days (after regression) and of pregnancy: 1-2, 3-4, 6-7 and >8 months. In these samples we investigated prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE) and their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS) and PTGE synthase (PTGES). The expression of mRNA was measured by RT-qPCR, hormones by EIA and localization by immunohistochemistry. The mRNA expression of COX-2 shows a clear regulation, with the highest level during early luteal phase (days 1-2) and than a significant decrease during mid luteal phase (P<0.05), with a slight increase during luteal regression (days >18). During pregnancy, the expression level was low agai. The mRNA expression PTGFS and PTGES in CL during early lutel phase (day 1-7) was high (P<0.05), followed by a continuous and significant down-regulation (P<0.05) to a lower plateau in CL during mid, late and regression as well as during all phases of pregnancy. The mRNA expression of PTGFR was high during all phases of estrous and pregnancy with prompt and significant downregulation (P<0.05) just during CL regression. In contrast, the mRNA of PTGER4 was low during all examined phases of estrous and pregnancy with clear significant upregulation (P<0.05) just during CL regression. The PTGER2 mRNA expression data showing no obvious differences in expression during different phases of estrous cycle and pregnancy. Immunohistochemically, the large granulose–luteal cells show strong staining for COX-2 and PTGES during early luteal stage, followed by lower activity afterwards. During pregnancy, most of the luteal cells were only weak positive or negative. In conclusion, our results lead to the assumption that the examined prostaglandin family members (COX-2, PTGFS, PTGES, PTGF, PTGE, and their receptors PTGFR, PTGER2, PTGER4) are involved in the local mechanisms regulating luteal function, namely during CL formation, function and regression and during pregnancy in the cow.

Use of Universal ProbeLibrary Probes for Detection of Highly Pathogenic Biological Agents

Karel Bílek, Kateřina Rosenbergová, Martina Grochová, Hana Placáková, Oldřich Kubíček, Jiřina Procházková

National Institute for NBC Protection (SÚJCHBO, v.v.i.), Czech Republic

In the world of the real-time PCR, there are two approaches how to obtain a fluorescent signal from the tube. Some investigators insist that unspecific intercalating dyes are a sufficient and cost-effective system to obtain a reliable signal. Others feel that only probe-based assays are the right approach. The UPL probes (Universal ProbeLibrary; Roche) offer a compromise that provides the specificity of a hydrolysis probe but does not require a unique probe for each target. The UPL probes use a nucleotide chemistry called LNA (Locked Nucleic Acid), which allows very short (8-9 bases) oligonucleotides to be effective in hybridization with the target. LNA allows the melting temperature of the short probes to be unusually high.

Hence, the objective is to demonstrate the use of the UPL probe for the detection of highly pathogenic biological agents. Moreover, we routinely combine the UPL probes with an intercalating dye (SYTO 61 dye; Invitrogen) for the verification of results. The detection strategy based on the use of the UPL probes was validated on various bacteria e.g. Brucella sp., Vibrio cholerae, Salmonella Typhi, Chlamydophila psittaci, further on selected Rickettsiae, as well as in the detection of viruses using RT-qPCR e.g. Chapare, Chikungunya, Ebola, Hantaan, Lujo, Seoul or Sin Nombre virus. Thanks to the shortness of the UPL probes sequences, it is possible to combine one UPL probe with multiple loci (in the combination with different pairs of primers). This feature was used for the detection of both Zaire and Sudan ebolavirus and for the detection of both the Brucella genus and Vibrio cholerae by the probes no. 138 and no. 11, respectively. All the presented assays were validated according to the MIQE guidelines.

In summary, the UPL probes can be used for the rapid, flexible and reliable detection and quantification of highly pathogenic biological agents. Furthermore, it is able to easily design gene-specific quantification assays using the UPL probes and the free web-based ProbeFinder software at the Assay Design

This work was supported by the Ministry of Interior of the Czech Republic no. VF20112015013 and VI20172019063

Using In Vitro Transcribed MRNA For Gene Therapy Targeting Viral Infections

Andreas Oswald, Ulrike Protzer

Institut für Virologie, Technische Universität München, Germany

Current gene therapy approaches mainly consist of lenti-, adeno- and adeno-associated viral vectors to introduce genes of interest into cells to reconstitute functionality of missing or defective factors. This approach has clear advantages, such as long-term gene expression after single administration or tissue specific delivery enabled by the tropism of the viral capsid, however there are safety concerns which complicate clinical translation. These include the risk of carcinogenesis arising from mutational integration, immune responses resulting from viral components or negative effects by long-term gene expression. Preexisting immunity against the used viral vector can render this approach ineffective. An approach to overcome these caveats can be the transfection of in vitro transcribed (ivt) messenger RNA (mRNA) that is transiently expressed in targeted cells or tissue. In contrast to viral vectors, this approach lacks viral components and the risk of unspecific integration into the genome.

One potential application is an mRNA based CRISPR/Cas9 system to address viral infections like chronic Hepatitis B by targeting the episomal viral genome (covalently closed circular DNA) or host factors involved in viral lifecycle. We produced ARCA capped Streptococcus pyogenes (Sp) Cas9 protein and ivt sgRNAs which target multiple sequences and genomic loci. In differentiated HepG2-NTCP, HepaRG and primary human hepatocytes mRNA transfection efficiencies were up to > 80 % with high cell viability (>95 %). Introduction of modified nucleotides (e.g. Pseudo-UTP, 5mCTP) significantly increased cell viability, protein expression and functionality. Targeting host genes (e.g. GAPDH or DNMT1) using SpCas9 mRNA showed genome editing that resulted in reduction of protein expression. Preliminary data in HepG2.2.15, a cell line harboring an integrated HBV genome, showed a significant reduction in HBe antigen expression 5 days after transfection of SpCas9 mRNA using sgRNAs against multiple loci in the HBV genome. Significant reduction of viral DNA expression after treatment with SpCas9 mRNA was detected via qPCR. In conclusion, our data indicate that mRNA-based gene therapy is a versatile tool for many in vitro approaches allowing fast and transient expression of SpCas9 or other proteins in dividing and non-dividing cells. Furthermore, ivt mRNA is a promising application for safe anti-viral therapy (as shown for HBV) that can be optimized for in vivo studies and clinical translation.

Whole Genome Crispr Screening to Identify Potential SERD Molecule Resistance Mechanisms

Christophe Marcireau, Karine Berthelot, Alice Williard, Hamida Fournet, Delphine Debono, Gilbert Thill, Helene Erasimus, Dorine Chassin, Christophe Lanneau, Veeranagouda Yaligara, Cecile Orsini, Michel Didier, Vincent Mikol, Monsif Bouaboula, Laurent Debussche

Sanofi, France

Metastatic breast cancer results in substantial morbidity and mortality for women afflicted with this disease. Approximately 80% of breast cancers express the estrogen receptor. The cancer cell survival and proliferation is driven by the activation of the estrogen receptor. SAR439859 is an effective endocrine therapy for breast cancer selectively and effectively degrading the estrogen receptor. A phase 1/2 study of SAR439859 alone and/or in combination with palbociclib in postmenopausal women with estrogen receptor positive advanced breast cancer was initiated last year by Sanofi.

CRISPR/Cas9 is a recent and revolutionary technology for efficient and directed alterations of the genome. CRISPR allows users to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs (SgRNA). These double strand breaks can be repaired by non-homologous end joining DNA repair mechanism. This DNA repair mechanism is an error prone DNA repair process which can lead to gene invalidation.

We aimed at investigating the potential SAR439859 resistance mechanisms. A better understanding of resistance mechanisms is needed to overcome this potential problem and to propose complementary therapeutic solutions. We conducted a whole genome CRISPR screening in a breast tumor cell line with a thermofisher commercial SgRNA library (ref: M04305) to identified genes involved in potential SAR439859 resistance phenomena. CRISPR screening steps will be presented from the cell line selections to results analysis.

6:00pm - 10:00pmENJOY: Welcome Reception
Lower & Upper Level 
Date: Tuesday, 19/Mar/2019
8:30am - 12:30pmdPCR: digital PCR
Session Chair: Jim Huggett, University of Surrey, United Kingdom
Session Chair: Afif Abdel Nour, Universite Saint Esprit de Kaslik, Lebanon (Lebanese Republic)
HS 14 

Biometrology, What Is It And What Could It Mean For Biomolecular Research?

Jim Huggett

University of Surrey, United Kingdom

While biomolecular methods are widely used to measure genes and gene expression, it is well documented that such approaches are often difficult to reproduce. So while I may be able to demonstrate a two-fold difference in the expression of a given gene, it can be very difficult for you to make that same measurement. Yet, unless the reason for this discrepancy is better understood the measurement I made may be difficult to corroborated and the chance of it becoming a useful biomarker reduced. There have been several initiatives among the molecular biology community to address this, such as MIQE, and increasing efforts amongst the in vitro diagnostic community to find solutions to assist in measurement standardisation. What is perhaps less clear amongst our community is the fact that there is an entire field of science dedicated to this very problem. Metrology is the scientific study of measurement and is applied in specialties such as physics to understand the accuracy of a measurement. This is achieved by determining mathematically how it is traceable to a given unit, which at its most accurate is to the Système international d'unités, or SI. By applying the concept to biological measurements, biometrology could enable us to understand why there are discrepancies between different laboratories as well as allow us to characterise sources of error and better understand the accuracy of a given measurement. This talk will explore how this new field of research is being applied to biomolecular measurement.

Molecular Characterization By ddPCR Of In Vitro Differentiated Oocyte-like Cells From Oogonial Stem Cells

Erica Silvestris, Paola Cafforio, Stella D'Oronzo, Claudia Felici, Francesco Silvestris, Giuseppe Loverro

University of Bari Aldo Moro, Italy

Recent reports regarding the presence of OSCs in the ovaries of non-menopausal and menopausal women suggest that neo-oogenesis is inducible during ovarian senescence. However, there isn’t consensus on isolation methods of these cells, their spontaneous maturation in vitro, and the final differentiation state of the resulting putative oocytes.

Ovarian cortex fragments from menopausal and non-menopausal women were processed by immuno-magnetic separation using a rabbit anti-human DDX4 antibody and cultured for up to 3 weeks. Large and small cells were individually isolated by DEPArray technology and early and late differentiation markers were measured by droplet digital PCR. The haploid versus diploid chromosomal content was investigated using fluorescence in situ hybridization (FISH).

After immuno-magnetic enrichment, DDX4-positive OSCs from non-menopausal and menopausal women, under appropriate culture conditions, differentiate into large haploid oocyte-like cells expressing the major oocyte markers growth differentiation factor 9 (GDF-9) and synaptonemal complex protein 3 (SYCP3) and then enter meiosis. Moreover, in culture small DDX4 positive cells are also present which do not express differentiation markers.

Therefore, we provide further evidence demonstrating the presence of stem-like cells with ovarian germ line properties within the otherwise exhausted oocyte reserve of menopausal human ovaries. These cells can be a source of oocytes that can be exploited to achieve fertility in women who are infertile or have an exhausted ovarian reserve.

Higher Order Multiplexing Using Digital PCR for CRISPR Gene Edit Validation

Alexandra Sarah Whale

LGC, United Kingdom

'Higher order multiplexing’ is the unique ability of digital PCR (dPCR) to precisely measure more targets than there are detection channels in the same reaction. In quantitative PCR, in order to measure three targets, three detection channels are required, however, in dPCR, the partitioning of the reaction into discrete partitions prior to the PCR enables three or more targets to be detected with two detection channels. This is achieved by varying the amount of primers and/or probes added to the reaction so that the end-point fluorescence is different between two targets with the same detection probe; the end-point fluorescence generates different “clusters” of partitions that can be observed on a 2D scatter plot. This talk will describe the different multiplex assay design strategies we have developed for genotyping and how precise and sensitive quantification can be achieved from counting the number of partitions in each of the clusters. The main example will illustrate our assay design for genotyping knock-in and knock-out CRISPR gene edits using a combined tandem probe binding and drop off probe design that we use to validate and determine the efficiency of desired gene edit.

Serial Flow Digital Droplet PCR, a New Paradigm!

Chris Perkins

Dropworks, Inc., United States of America

DropWorks introduces FluxPCR™; a fast and flexible platform for performing digital assays in self-contained flowing droplets. Aiming to help users transition from qPCR to digital PCR, DropWorks has reduced both the cost and complexity required to generate high quality digital PCR data. A single touchpoint workflow reduces error and serial sample processing provides the flexibility to run only a few samples or an entire plate without wasting expensive consumables. An instinctive user interface and software streamlines data production and ensures that users can spend less time processing their data and more time understanding what that data means for the underlying science.

Eight Years Later This Is What Digital PCR Offered To Science

Afif Abdel Nour

The Holy Spirit University of Kaslik, Lebanon

Throughout the past 36 years Polymerase Chain Reaction (PCR) has proven to be the most powerful technique in a scientist toolbox. The overwhelming evolution from the first generation (PCR) to the “Gold standard” second generation (Real Time quantitative PCR A.K.A. qPCR) and recently to the third and breathtaking generation the digital PCR (dPCR), proven that DNA/RNA amplification will always be a popular method.

To understand the use of digital PCR, the Scopus database was screened for the terms “digital PCR, ddPCR and dPCR”, in all papers that were published between 2011 and 2018. We considered only papers published in journals and we excluded books, books chapters and conferences. Then we classified those papers by country, subject areas, CiteScore, institutions... The aim of the current analysis is to shed the light on the good and bad students in terms of usage of the dPCR and the citation of the digital MIQE guideline. We than studied the impact of dPCR on different research area like oncology, single cell analysis and diagnostics.

Highly Multiplexed Detection of GMOs in Food Samples by Crystal Digital PCR

Katharina Lührig1, Caroline Charky2, Maximilian Neugebauer1, Heike Ziebarth1, Kornelia Berghof-Jäger1

1BIOTECON Diagnostics GmbH, Germany; 2Stilla Technologies, France

Stilla Technologies will present the latest technical updates of the NAICA system along with the outcome of a cooperation between Stilla Technologies and BIOTECON Diagnostics to deliver a complete solution for the detection of soy GMOs in food samples.

As the number of authorized GMOs in the European market is increasing, faster and cost-efficient detection methods are needed. We present digital PCR as a suitable alternative to real-time PCR which allows multiplexed GMO quantification without the need for a standard curve. A new digital PCR assay developed by BIOTECON Diagnostics on the NAICA system allows the quantification of all 14 soybean GMO events currently authorized in the EU in just a single reaction. This assay reliably and precisely quantifies GMO contents at the regulatory thresholds of 0.9% and 0.1%. A summary quantification of all authorized soy events present in a sample is provided making quantification with single assays dispensable. We will highlight the particular advantages of the Crystal Digital PCR platform for R&D and routine analytics.

Resolving Translational Oncology Research Challenges with NanoString Direct Digital Gene Expression Analysis.

Christoph König

Nanostring Technologies, United Kingdom

Successful Translational Research and Biomarker development today much depends on the availability of technologies that ensure accurate and precise multiplexed detection and quantitation of target biomolecules. Clinical necessities, on the other side, dictate that such measurements can be done effectively on small sample input amounts, on material of poor starting quality, and analytes obtained in a minimally invasive way. Because of this, classical methods often encounter problems to measure RNA or Protein expression levels robustly and reproducibly at high multiplexing levels.

The NanoString digital gene expression technology overcomes these hurdles by offering a fast workflow that is just based on hybridization and does not require any enzymatic modification of analytes or library preparation. The talk will present achievements of the NanoString approach in the context of biomarker discovery and pathway analysis within various areas of cancer research. It will also show how the technology can be employed to resolve expression differences in tissue sections in the spatial dimension.

8:30am - 12:30pmLB: Liquid Biopsy
Session Chair: Jo Vandesompele, Ghent University & Biogazelle, Belgium
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
HS 15 

Cell-Free Tumor DNA Analysis In Liquid Biopsy Using Ultrasensitive Sequencing

Anders Ståhlberg1,2

1University of Gothenburg, Sweden; 2Sahlgrenska University Hospital, Sweden

Liquid biopsy and detection of tumor associated-mutations in cell-free circulating DNA (ctDNA) often requires the ability to identify single nucleotide variants at allele frequencies below 0.1%. Standard sequencing protocols cannot achieve this level of sensitivity due to background noise from DNA damage, polymerase induced errors. Addition of unique molecular identifiers allows identification and removal of errors responsible for this background noise. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. We show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. Theoretically, high fidelity enzymes will reduce error rates in barcoded NGS but this has not been thoroughly explored. We evaluated the impact of polymerase fidelity on the magnitude of error reduction at different steps of barcoded NGS library construction. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.

Clinical Application of Liquid Profiling for Precision Medicine

Stefan Holdenrieder

German Heart Center Munich, Germany

The concept of “precision medicine” in the treatment of cancer patients aims to specifically target deregulated molecular cancer pathways that are involved in cancer cell proliferation, angiogenesis, metastasis and evasion of the immune control. Classical ways are extracellular blockage of membrane-bound growth receptors by specific antibodies or intracellular inhibition of cancer pathways by small molecules like tyrosine kinase inhibitors that have shown promising therapeutic results. New approaches aim to restore and reactivate immune cell functions to attack the tumor in a more sustainable way. Precondition for precisely acting drugs is the presence of the respective molecular changes that have to be detected in tumor tissue or in the blood plasma – also known as “liquid biopsy” or “liquid profiling” – prior to therapy.

Sensitive blood-based diagnostics are able to identify druggable mutations in cell-free plasma tumor DNA (ctDNA) and circulating tumor cells (CTC). Current diagnostic strategies include single-, multi-gene and whole exome / genome approaches that have recently become more sensitive and specific by the introduction of tumor-enrichment and error-reducing techniques. As liquid profiling is only minimally invasive it can be used to complement tissue biopsy for patient stratification and for the serial monitoring of successfully treated and newly occurring resistant cell clones at an individual level.

With only few exceptions, plasma ctDNA is measurable in patients with most tumor types, particularly at advanced stage of disease. Concordance with tumor tissue is around 90% if highly sensitive methods are used. ctDNA diagnostics support therapy stratification if tumor biopsy is not available or insufficient, has predictive and prognostic power and can be used as modern, quantifiable tumor marker for monitoring mutation-positive patients. Finally, multiplexing and sequencing enables the detection of new mutations. Preanalytics and rigorous quality control have turned out to be critical for reproducible results. Today, the use of blood conserving tubes, double centrifugation, standardized DNA extraction, enrichment, quantification and sequencing techniques are basic requirements for ctDNA diagnostics.

Plasma-based ctDNA “companion diagnostics” has developed to a valuable new tool for precision medicine. First assays are available as IVD-CE labelled methods to be applied in routine diagnostics. High grades of technological and quality standards as well as future combination with protein and metabolome markers will help to improve the diagnostic accuracy and facilitate new applications in other fields of precision medicine such as in immune checkpoint therapies.

Exploiting RNA in Liquid Biopsies for Precision Medicine Purposes

Jo Vandesompele1,2

1Ghent University, Belgium; 2Biogazelle, Belgium

In contrast to general belief, a substantial part of the human transcriptome is abundantly present in the blood and other biofluids as extracellular messenger RNA, long non-coding RNAs and various small RNAs, ready to exploited. I will discuss various workflows for RNA sequencing of biofluid derived RNA, including probe-based target capture and unbiased total RNA library prep as sensitive RNA sequencing workflow to study thousands of mRNA and lncRNA genes in cell-free RNA from patients’ plasma and other biofluids. Apart from RNA abundance profiling, this type of data can also be used to detect structural RNA variants, such as somatic mutations, fusion genes and RNA editing events, all known to play an important role in disease, including cancer. The resulting RNA profiles can be deconvoluted to enumerate the cells, tissues and organs that contribute to the extracellular RNA. Human biofluid RNA sequencing enables liquid biopsy guided precision oncology, such as therapy stratification, treatment response monitoring and early detection of relapse. I will also discuss the pre-analytical jungle of RNA targeted liquid biopsies and need for standardization, as part of the ongoing extracellular RNA quality control study. I will end with the first insights of the Human Biofluid RNA Atlas, in which we have deeply probed into the extracellular transcriptome of 22 human biofluids, providing a solid foundation for exploiting biofluids for diagnostic purposes. (on behalf of Extracellular RNA Quality Control consortium, Human Biofluid RNA Atlas consortium).

Liquid Biopsy -- Exosomal microRNA Biomarker Signatures in Clinical Diagnostics.

Michael W Pfaffl

Division of Animal Physiology & Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Germany

Extracellular vesicles (EVs) circulate in body liquids and are involved in the intercellular communication. They have important regulative functions in almost any physiological or pathological process. In recent time especially the exosomes have gained huge scientific interest because of their molecular diagnostic potential, mainly based on the containing microRNA pattern. The past decade has brought about the development and commercialization of a multitude of extraction methods to isolate EVs and exosomes, primarily from blood compartments. The exosome purity and which subpopulations of EVs are captured strongly depend on the applied isolation method, which in turn determines how suitable resulting samples are for potential downstream applications and biomarker discovery. Herein we compared the overall performance of various optimized isolation principles for serum EVs/exosomes in healthy individuals and critically ill, mainly pneumonia and sepsis patients. The isolation methods were benchmarked regarding their suitability for biomarker discovery as well as biological characteristics of captured vesicles, according to the latest MISEV 2018 guidelines. To analyze the small-RNA deep sequencing results, a self-established bioinformatics pipeline for microRNA (based on R) and a deeper analysis of their isoforms (via isomiRROR) was applied.

Final goal was the development of microRNA/isomiR biomarker signature for an early diagnosis and a valid classification of critical ill patients. Various patient cohorts were investigated: healthy volunteers, sepsis (referred to mild or severe pneumonia), acute pulmonary failure (ARDS) and septic shock. Distinct miRNA signatures were identified, which are applicable to indicate disease progression from limited inflammation present in pneumonia to severe inflammatory changes as seen in ARDS or sepsis shock. The study results indicate that EV miRNA biomarkers have future potential for early diagnosis of pneumonia and to indicate disease progression towards severe inflammation events. Further the methodological findings provides guidance for navigating the multitude of EV/exosome isolation methods available, and helps researchers and clinicians in the field of molecular diagnostics to make the right choice about the EV/exosome isolation strategy.

New Developments in Sample Quality Control - Focus on Cell-free DNA

Bettina Strauch, Elisa Viering

Agilent Technologies, Germany

Quality control of nucleic acid starting material is essential to ensure the success of downstream experiments. Especially, Next Generation Sequencing (NGS) developed to a powerful tool in almost all genetic research and diagnostic areas. Due to the establishment of low input library protocols for NGS workflows sequencing of cell-free DNA (cfDNA) became possible. Since the downstream applications are often time-consuming and expensive, tight QC steps are required to avoid a “garbage in-garbage out” situation.

This talk focuses on standardized nucleic acid quality assessment using different automated electrophoresis platforms to ensure that samples are “fit for purpose”. Accurate quantification of starting material (DNA, RNA, cfDNA, FFPE samples) is essential to determine suitable input amounts for library preparation prior to sequencing. Depending on preanalytical sample treatment or extraction methods the quality of e.g. cfDNA can vary. This results in various electropherogram patterns after electrophoretic separation as presented in this talk. Moreover, the electrophoretic separation enables to qualify cfDNA samples according to their contamination level with high molecular weight material. Likewise, quality scores for gDNA, RNA as well as FFPE RNA can be assessed using automated electrophoresis systems, which allow defining a quality threshold for specific types of samples or preparation. This allows defining thresholds for objective sample qualification prior to library preparation.

Exosome-derived Epigenetic Biomarkers for Saliva Diagnostics

Ulrike Kegler1, Manuela Hofner1, Anja Buhmann1, Helene Scharkosi1, Walter Pulverer1, Michael Leutner2, Klemens Vierlinger1, Alexandra Kautzky-Willer2, Christa Nöhammer1

1AIT Austrian Institute of Technology GmbH, Molecular Diagnostics, Vienna, Austria; 2Medical University Vienna, Division for Endocrinology and Metabolism, Vienna, Austria

The aim of our research activities at AIT, the Austrian Institute of Technology, is to define reliable biomarkers suitable for early and non-invasive disease diagnosis and prognosis. To this end we have been establishing and optimizing a whole range of multiplex capability technologies (e.g. microarrays, quantitative PCR, Luminex bead technology) to meet the special demands and challenges of diagnostic biomarker discovery - and validation in body fluids. Using this specific technology expertise we e.g. successfully discovered autoantibody- as well as DNA methylation -based diagnostic marker panels for the big 4 cancer entities (breast, colon, prostate, lung) in serum or plasma. Based on these success stories and the evident advantages of saliva as a diagnostic matrix our recent special interest is to go for saliva diagnostics and to evaluate saliva for its suitability for circulating biomarker-based diagnostics. Along these lines we will show proof of concept studies for autoantibody- and DNA-methylation based salivary diagnostics and report on the evaluation of different commercially available strategies for isolation of exosomes from human serum and saliva. We will further present data from comparative profiling studies in salivary - and serum-derived exosomes including targeted protein-, genome-wide microRNA – as well as DNA-methylation profiling. Last but not least we will report on first results of a research project where we are looking for salivary and plasma exosome-derived epigenetic biomarkers for early type 2 diabetes diagnosis.

Sensitive, Easy And Fast Monitoring Of Treatment Efficiency, Resistance Development And Disease Progression Using Open Platform QPCR On Liquid Biopsies From Cancer Patients

Ulf Bech Christensen, Rasmus Koefoed Petersen

PentaBase, Denmark

Personalized administration of present and future targeted cancer therapies demands fast, low cost and easy-to-use companion diagnostics (CDx) providing doctors and patients with the answers they need to maximize treatment efficiency and minimize treatment costs. We have developed and clinically validated several real-time PCR based assay for liquid biopsies. Sensitivity studies show that our SensiScreen® Liquid assays can detect down to a single copy of mutated DNA in a background of wild type plasma cell-free DNA (cfDNA) in a simple and robust workflow.

The extreme sensitivity of our assays is obtained by use of BaseBlockers™, that suppress amplification of wild type DNA while allowing amplification of mutated DNA, modified primers and sensitive dual-labelled Probes. BaseBlockers™, primers and probes are all based on the DNA platform technology – Intercalating Nucleic Acid, INA®.

We will present a case study using our SensiScreen® Liquid CE IVD Assay for monitoring disease progression and treatment efficiency in a clinical setting, in a BRAF V600E mutated metastatic colorectal cancer patient. After initial diagnosis and genotyping, we have monitored the patient’s cfDNA levels and the cfDNA amount of BRAF V600E in plasma for more than 700 days during three different regimes of treatment and compared with carcinoembryonic antigen and CA 19-9 levels.

We believe that sensitive monitoring of liquid-based biopsies present unique opportunities for healthcare providers, targeted therapy developers, and eventually cancer patients. The information and additional knowledge gained on patient response and efficacy of treatment can be used as a tool to reduce use of inefficient treatment and improve clinical output.

12:30pm - 2:00pmJournal: BDQ Editors Meeting
Session Chair: Jim Huggett, University of Surrey, United Kingdom
Session Chair: Justin O'Grady, Quadram Insitute Bioscience, United Kingdom
HS 15 
12:30pm - 2:00pmPO2: Tuesday Lunch Poster Session
All posters will be displayed in parallel at all three poster sessions PO1, PO2 & PO3 => the poster viewing can be done from "Monday Evening Main Poster Session" till "Wednesday Lunch Poster Session"
Lower Level 
12:30pm - 2:00pmLU2: Lunch Tuesday
2:00pm - 6:00pmAMDx1: Advanced Molecular Diagnostics 1
Session Chair: Carl T Wittwer, University of Utah, United States of America
Session Chair: Mikael Kubista, TATAA Biocenter AB, Sweden
HS 14 

Rapid qPCR and the Future of Molecular Diagnostics

Carl T Wittwer

University of Utah, United States of America

We experimentally dissected the kinetic requirements of PCR into the stages of DNA denaturation, primer annealing, and polymerase extension. A commercial capillary LightCycler™ was used to correlate PCR product length to required extension times. More accurate results for all stages were obtained by using a custom “extreme” temperature cycling instrument. Increased primer and polymerase concentrations were allowed to increase speed, and actual temperatures were measured in real time rather than programmed or predicted. Two stages were left long and not limiting, while the other was varied until Cqs increased. Under the conditions used, polymerase extension times depended on product length with about 1 s needed for every 100 bps. Cqs started to increase after 200-500 ms above the denaturation threshold, and after 300-1,000 ms below the annealing threshold. Temperature thresholds were set by including >98% of the derivative melting curve areas as experimentally determined by melting curves under PCR conditions. Progressing from rapid cycle PCR to extreme PCR has decreased cycling times by 10-60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.

Impact of Endogenous and Exogenous Alterations of Glucocorticoids on the Derangement of Clock-related Genes in Peripheral Mononuclear Cells

Mary Anna Venneri

Sapienza University of Rome, Italy

Circadian clock genes regulate the physiological sensitivity to rhythmic release of glucocorticoids (GCs). In turn, GCs have reciprocal effects on circadian system. Conditions affected by hyper- or hypocortisolism are characterized by a loss of circadian rhythmicity. Loss or impairment of circadian rhythms have been reportedly associated with malignancies and an increased incidence of infectious diseases, suggesting a role of immune function alterations. Adrenal insufficiency (AI) requires life-long glucocorticoid replacement. Conventional therapies fail to mimic the endogenous cortisol circadian rhythm. Objective of this study was to evaluate the effect of the timing of GC administration on circadian gene expression in peripheral blood mononuclear cells (PBMCs) of AI patients from DREAM trial. Patients were randomly assigned to continue their multiple daily doses, or switch to an equivalent dose of once-daily modified-release hydrocortisone, and compared to healthy controls. Circadian genes in PBMC were analyzed by quantitative by real-time qRT-PCR using predesigned 96-well panel SYBR® Green Circadian rhythms (SAB Target List) H96 (Bio-Rad, PrimePCR®). Compared to healthy controls, 19 of the 68 genes were found differentially expressed in AI patients at baseline, 18 of which were restored to control levels 12 week after switching therapy, comprising: ARNTL, CLOCK, PER3, TIMELESS, AANAT, CAMK2D, CREB1, CREB3, MAPK1, WEE1, PRF1. Changes in WEE1, PRF1 and PER3 expression correlated with change in clinical outcomes including glycated hemoglobin, inflammatory monocytes and CD16+ NK cells, suggesting that the extent of reprogramming of circadian gene expression can be linked to the magnitude of improvement in clinically measurable outcomes. In conclusionAIpatients on standard therapy exhibit a dysregulation of circadian genes in PBMCs. The once-daily administration reconditions peripheral tissue gene expression to levels close to healthy controls, and correlates with clinical improvement.

SD DNA Polymerase: A New Tool for Variety of Molecular Biology Applications

Konstantin Ignatov, Andreas Kirsten, Vladimir Kramarov, Ferdinand Holzinger, Sergey Kovalenko

Bioron, Germany

Till recently DNA polymerases were either suitable for PCR amplification or isothermal amplification, but not both. The new SD polymerase (Bioron GmbH Patent US 9,896,671) is a Taq DNA polymerase mutant that was created as a result of successful attempt to combine the thermostability and robust polymerase activity of Taq DNA polymerase with strong strand displacement activity of Bst DNA polymerase. Currently SD polymerase seems to be the only enzyme suitable for PCR which possesses strong strand-displacement activity. New polymerase was shown to be suitable in the number of unique applications such as heat pre-denaturated LAMP and PCDR (Polymerase Strand Displacement Reaction). SD polymerase appeared to be effective in conventional PCR, Long-PCR and amplification of GC-regions with complex secondary structures. Moreover, SD polymerase can be effectively used in WGA (Whole Genome Amplification) based on DOP-PCR, in the Single Cells WGA, in the new NGS-library construction methods and in the newly developed template-independent tailed tandem repeat PCDR (TTR-PCDR).

Since SD-polymerase allows performing Strand Displacement and PCR in the same reaction, the work flow for the SD polymerase based techniques are usually comfortable, simple and friendly.

New techniques based on the advantageous properties of SD polymerase are currently under development by several biotech companies over the world. Thus, SD polymerase is able to improve the existing DNA amplification techniques and can be used for creation of new convenient methods of DNA manipulations.

Methylation Sensitive High Resolution Melting; A Sensitive And Specific Method For High-Throughput Assessment Of Methylation.

Massimo Santoro

IRCCS Fondazione Don Carlo Gnocchi, Italy

DNA methylation is an epigenetic mechanism which implies heritable changes of gene expression without a change in the primary DNA sequence. Covalent histone modifications and methylation changes of cytosine at CpG dinucleotides are the most widely investigated epigenetic mechanisms. DNA regions with a relatively high CpG dinucleotide content are referred to as CpG islands that are distributed in a non-random manner across the human genome and often span 5’ untranslated region (UTR), 3’ UTR, promoter region and the first exon of protein coding genes. Methylation of CpG islands usually acts to turn off (silence) transcription by recruiting histone deacetylases thereby inducing the formation of inactive chromatin. Mapping of methylation patterns in CpG sites is an important tool for understanding both normal and pathogenic gene expression events.

Numerous technique are used for detection of CpG methylation, among them, PCR-based protocols are most widely used. Because PCR amplification removes methylation marks, the DNA template is chemically modified by sodium bisulfate that converts all unmethylated cytosines to uracil, leaving methylated cytosines unaltered and preserving methylation information before PCR amplification.

Subsequent amplification of bisulfite-modified template results in different amplicons from methylated and unmethylated templates with different melting profiles when subjected to thermal denaturation.

The methylation-sensitive high resolution melting (MS-HRM) technology is based on the comparison of the melting profiles of sequences that differ in base nucleotide composition. The PCR product originating from the methylated allele will have different GC content from PCR product derived from unmethylated variant of the same locus. MS-HRM allows for estimation of the methylation level by comparing the melting profiles of unknown PCR products to the melting profiles of PCR products derived from standards with a known unmethylated to methylated template ratio (range from 0 to 100 methylation percentage).

Here, we show the application of MS-HRM in two different studies:

1) detection of methylation levels in the 3’UTR of dystrophia myotonica protein kinase (DMPK) gene in a cohort of 66 myotonic dystrophy type 1 (DM1) patients (age 38.6±12.5 years) and 30 age-matched healthy controls (age 40.3±13.8 years).

2) detection of promoter methylation levels in cannabinoid type 1 (CNR1) and 2 (CNR2) receptors of 12 multiple sclerosis secondary progressive (MSS-SP) patients (age 54.2±11.7 years) before and after treatment with Sativex®.

Both papers show how the MS-HRM protocol provides a high-throughput platform for cost- and labor-efficient screening for methylation changes. Moreover, the simplicity and high reproducibility of this technique makes MS-HRM the method of choice for methylation assessment in both research and diagnostic applications.

Two-Tailed Quantitative PCR for Precison Diagnostics

Mikael Kubista1,2, Robert Sjöback1, Andrei Herdean1, Peter Androvic2, Lukas Valihrach2

1TATAA Biocenter AB, Sweden; 2Institute of Biotechnology, Czech Academy of Sciences

We present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on novel chemistry called Two-tailed PCR. Two-tailed PCR takes advantage of target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of 7 logs and a sensitivity sufficient to detect less than ten target miRNA molecules. The reverse transcription step can be multiplexed and it allows for rapid testing with a total analysis time of less than 2.5 hours. Several applications will be presented including a quality control panel for liquid biopsy samples and 1-tube combined analysis of mRNA and microRNA.

P. Androvic, L. Valihrach, J. Elling, R. Sjöback, M. Kubista. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Research, Volume 45, Issue 15, 6 September 2017, Pages e144

Precision Liquid Biopsy Based Nucleic Acid Based Molecular Diagnostics Powered by Xenonucleic Acids

Michael Joseph Powell

DiaCarta, Inc., United States of America

Current clinically available molecular tests for detection of pathogenic nucleic acid variations especially tumor derived oncogenic 'driver' and drug resistant somatic mutations that are performed on circulating cell-free nucleic acids present in biological fluids such as patient’s blood plasma have limited sensitivity. This is because of the low frequency of these gene variations and the large excess of wild-type nucleic acids present. In order to achieve high sensitivity for the detection of only a few target molecules (mutant alleles) present in a vast excess of non-target molecules (wild-type alleles) sophisticated methodologies that require expensive instrumentation, highly skilled operators and in some cases intensive computational bioinformatics methods such as digital-droplet PCR (ddPCR), BEAMing PCR and next generation deep sequencing (NGS) are being employed in large clinical research centers. The limited availability, high cost and long analysis times of these methods prompted us to develop a new technology that can be performed globally by existing pathology personnel with instrumentation that is already present in every hospital pathology laboratory. At the heart of this innovative technology are novel molecular nucleic acid analogs that we call xenonucleic acids (XNA) that possess all the natural bases that occur in DNA appended to a new chemical backbone that imbibes these oligomeric nucleic acid binding molecules with exquisite specificity and high binding affinity for complementary target sequences. Any variation in the sequence that the XNA binds to creates a differential binding phenomena that can be exploited to develop real-time qPCR and extremely high sensitivity NGS assays that can detect as little as 2 copies of variant templates in a large excess of wild-type templates in DNA obtained from tissue biopsies or more preferably plasma. Commercial CE/IVD Certified Products have been developed and validated that include QClampTM gene specific real-time qPCR based tests, a new highly sensitive blood-based colorectal cancer detection test called ColoScapeTM and a high sensitivity targeted amplicon based target NGS platform called OptiSeqTM. This presentation will discuss the new technology and the improved and widely available opportunities that it affords for improved precision diagnostics and targeted therapies of human diseases particularly cancer.

Multiplex xMAP Technology for a Complex Detection of Pathogenic Agents Significant for the Protection of Human and Animal Health

Pavlina Jelinkova, Petr Kralik

Veterinary research institute/Brno, Czech Republic

Early detection of pathogenic agents in the human and veterinary field is one of the main and key moments for the treatment of infectious diseases. For this we use molecular biological methods, such as a polymerase chain reaction (PCR). Methods of molecular diagnostics are generally focused on finding specific or virulence genes and therefore the subsequent design of probes which are necessary for detection of the selected pathogen. Also specific mutation in gene, or single nucleotide polymorphism (SNP) can be used in probe design. Nowadays, a large number of pathogens need to be identified during one reaction in a short time. One of the possible multiplex sample analysis is xMAP technology (x = analyte, MAP = Multi Analyte Profiling). xMAP technology is based on a combination of existing laboratory methods such as PCR, flow cytometry and ELISA, and enables detection of more than 50 different analytes (nucleic acids) simultaneously during one reaction. In this case, a multiplex oligonucleotide ligation (MOL) is performed prior to the PCR in which there is only one pair of universal primers. One of the primers is labeled with a fluorescent dye. xMAP technology uses magnetic microspheres with a special spectral address, to which the analyte is then binds specifically with the sample.

In our laboratory, we focused on obtaining a comprehensive protocol for the MOL-PCR method followed by MagPix analysis. The assay is suitable for rapid multiplex detection of bacteria, parasites and also viruses in real samples. The final result of this work is the creation of individual multiplexing systems (detection panels). At present, we have developed diagnostic panels for the multiplex detection of 5 bacteria (Campylobacter jejuni, Escherichia coli – EHEC, Yersinia enterocolitica, Listeria monocytogenes, Salmonella enterica) and 4 parasites (Toxoplasma gondii, Taenia saginata, Trichinella spiralis, Giardia intestinalis). We continue to develop a panel for identification of pathogens that can be used in bioterrorism: Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis.

This work was supported by Security Research of Ministry of the Interior of the Czech Republic VI20152020044.

2:00pm - 6:00pmISEV: ISEV Satellite Session
Session Chair: Kenneth Witwer, The Johns Hopkins University School of Medicine, United States of America
Session Chair: Hidetoshi Tahara, Hiroshima University, Japan
HS 15 

Mesenchymal Stem Cell-derived Extracellular Vesicles: a Potential New Tool in Regenerative Medicine

Bernd Giebel

Institute for Transfusion Medicine, Germany

Human mesenchymal stem/stromal cells (MSCs) represent a promising tool in regenerative medicine. Up to now, more than 800 NIH-registered clinical trials investigated their immunomodulatory and pro-regenerative therapeutic potential in various diseases, including graft-versus-host disease (GvHD) and ischemic stroke. Despite controversial reports regarding the efficacy of MSC-treatments, MSCs seem to exert their beneficial effects rather in a paracrine manner than by cell replacement. In this context, extracellular vesicles (EVs), such as exosomes and microvesicles, are discussed to execute the MSCs’ therapeutic effects. Indeed, we observed beneficial therapeutic impacts of MSC-EVs in a patient, who suffered from steroid-refractory acute GvHD. Furthermore, beneficial effects were observed in animal models for several different diseases.

According to controversial reports in the MSC field, especially since a phase III clinical trial failed to show clinical efficacy in MSC treated GvHD patients, we have started to compare immunomodulatory effects of independent MSC-EV preparations. Indeed, in our in vitro assays independent MSC-EV fractions reveal different immunomodulatory capabilities. To unravel the basis for these differences we are currently using several methods to dissect the heterogeneity between and within given MSC-EV samples.

Standardization in EV and exRNA Research: MISEV and Beyond

Kenneth Witwer

The Johns Hopkins University School of Medicine, United States of America

Rapidly growing interests in physiologic and pathologic processes associated with extracellular vesicles (EVs), including presumed functions of various EV RNA molecules, have created a need for standardization initiatives to shape and guide the field. As an example, the International Society for Extracellular Vesicles (ISEV) first published the Minimal Information for Studies of Extracellular Vesicles (“MISEV”) in 2014 to call attention to rigorous EV characterization approaches and experimental controls. In this presentation, I review recent developments in EV and exRNA standardization, focusing especially on the “MISEV2018” guidelines in the Journal of Extracellular Vesicles. These guidelines were prepared with methods for broad community input and buy-in that may serve as a model for ongoing and future initiatives. We will discuss outstanding needs in the field and what might be done to address them.

Circular RNAs In EVs - A Novel Class Of Released Non-coding RNAs

Christian Preußer

Justus Liebig University of Giessen, Institute of Biochemistry

Non-coding RNAs (ncRNAs) were established in the last decade as a new valuable biomarker class for human diseases. Specifically, circular RNAs (circRNAs), which represent the newest class of ncRNAs, turned into research focus within the last decade. Although single examples of these particular RNA class had been known for more than forty years, circRNAs were established as a large RNA class only a few years ago, based on the advent of tailored RNA-Seq technologies and Bioinformatics. CircRNAs were identified in all eukaryotic cells investigated so far, are cell-type specifically expressed, and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form, resulting in a metabolically more stable form of RNA compared to their linear counterparts. These biological properties and the correlation with various human diseases, such as cardiovascular diseases and cancer, immediately suggesting their potential use as novel attractive RNA-based biomarkers.

We have focused on the analysis of circRNAs and the corresponding linear splice isoforms from human blood cells, in particular platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles (EVs) from purified and in vitro activated human platelets, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. Furthermore, to unravel mechanisms that contribute to the specific packaging of RNAs into EVs, we developed a novel approach to determine specific sequence motifs required for selective loading of (circ)RNA into EVs. This unbiased method should contribute to our understanding of how RNAs are specifically packaged into EVs.

In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and harbor great biomarker potential. In addition, we would also like to stress the strong need for standardization and for setting up minimal requirements for validating this class of ncRNA.

Characterization of Extracellular vesicles (EVs) Derived ExRNA in Cancer Using NGS

Yukie Nishiyama1, Makoto Tahara2, Hidetoshi Tahara1

1Department of Cellular and Molecular Biology,Institute of Biomedical & Health Sciences, Hiroshima university, Japan; 2Department of Head and Neck Medical Oncology, National Cancer Center Hospital East, Kashiwa, Japan.

MicroRNAs (miRNAs) are small non-coding RNAs approximately 21 nucleotides in length that regulate posttranscriptional gene expression. miRNAs exist in exosomes, which are 50–100nm in size extracellular vesicles (EVs) and secrete from mammalian cells. Recent studies have demonstrated that miRNAs and other small RNAs stably exist in body fluids and their expression patterns in cancer patients are distinct from those in healthy individuals. In this study, we analyzed non-coding small RNAs including miRNAs that specifically exist in plasma/serum of patients with breast and tongue cancer by using NGS plat form. We identified a number of biomarker candidates such as mature miRNAs, isomiR, tRNA-derived fragments (tRFs), and other ncRNA, are known to regulate expression of genes involved in cell metabolism and are released into body fluid from various cells with extracellular vesicles. To test the possibilities that these candidate small RNAs are secreted from cancer cells, we purified EVs from serum in health volunteer and cancer patients, and analyzed small RNAs using NGS, and found that some of candidate small RNAs can be found in EVs. Interestingly, these small RNAs are also found in culture media in cancer cell lines. Based on our alogism, we identified significant biomarker secreted from cancer cells for early detection of cancer.

Distribution Of Non-coding RNA Types Over EV And Other RNA Carriers In Plasma

Esther Nolte-'t Hoen, Tom Driedonks, Susanne van der Grein

Utrecht University, The Netherlands

EV contain RNAs, lipids, and proteins and act as multicomponent signaling entities during intercellular communication. The release and composition of EV depends on the activation/differentiation status of EV-producing cells.

The currently most intensely studied EV-RNA biotypes are miRNAs and mRNAs, some of which have been implicated in disease progression and/or proved valuable as biomarkers. However, we previously showed that EV released by many different cell types are particularly rich in other small RNA biotypes such as tRNA, Y-RNA, snRNA, and snoRNA, which may also exert gene regulatory functions. Examples will be shown on how environmental triggers can lead to changes in the type of released EV and their small non-coding RNA content. Levels of not only miRNAs, but also other non-coding RNA types varied depending on the status of the parent cell and these may be further explored as biomarkers or functional entities within EV.

RNA in plasma is not only enclosed in EV but can also be associated to other macromolecular structures, such as ribonucleoprotein particles (RNPs) and lipoprotein particles. These structures overlap in size and/or density with EV and may be co-isolated in commonly used EV isolation procedures, such as ultracentrifugation. By using several separation techniques and protease/RNase treatment strategies, we show how different miRNAs and other non-coding RNAs are distributed over various carrier structures in plasma. In-depth characterization of these RNA carriers and changes in their RNA content induced by cell activation or disease is important to further elucidate the function and biomarker potential of the different extracellular RNA types.

Normalizing Urinary Extracellular Messenger RNA Biomarkers: Theoretical Considerations and a Review of Experimental Findings

Pradeep Moon Gunasekaran1, J. Matthew Luther2, J. Brian Byrd1

1University of Michigan Medical School, United States of America; 2Vanderbilt University School of Medicine, United States of America

Messenger RNA (mRNA) has been extensively annotated, and its crucial role in the central dogma has made it a key target in many studies of biomarkers and drug targets. Extracellular vesicles shuttle mRNA, among other molecular cargo. mRNA in urinary extracellular vesicles has potential as a biomarker, particularly in diseases affecting cells of the urothelial tract. There is evidence that this mRNA could provide information about transcription in cells of urogenital tissues. However, the optimal means of normalizing these signals is unclear. In the more common cell lysate context, gene expression can be normalized to robustly expressed genes that have similar expression between cells. However, in the context of extracellular mRNA shuttled by extracellular vesicles, normalization strategies remain undefined.

This talk will describe relevant first principles as well as research findings from our lab and other labs toward normalization of urine extracellular mRNA. The talk will focus on at least two hypothetical sources of confounding that might be important denominators when normalizing assays of urinary extracellular mRNA transcripts. The first is changes in the composition of the biofluid matrix broadly affecting analytes’ concentrations (i.e., intra-individual changes or inter-individual differences in urine composition due to having recently consumed water). The second is broad changes or differences in the expression of genes without direct relevance to the specific biology of interest. The extent to which each of these factors must be taken into account during normalization will depend on the magnitude of target gene’s expression signal in the disease relative to the magnitude of the noise created by these potentially confounding factors. The rationale for the use of urinary creatinine as the traditional reference molecule for many urinary assays will be discussed, as will advantages and disadvantages of urinary creatinine for normalization of urinary extracellular mRNA. In addition, the possibility of using reference genes to normalize assays of urinary extracellular mRNA will be discussed. Prior experiments bearing on the feasibility of these approaches will be reviewed. The talk will also discuss other potential strategies for normalization, such as normalizing to the concentration of extracellular vesicles. Finally, the talk will attempt to synthesize these ideas and the findings from the literature into suggestions for those developing urinary extracellular mRNA assays.

Serum-Free Media Supplements Carry MiRNAs That Co-Purify With Extracellular Vesicles

Martin Auber, Dominik Fröhlich, Krämer-Albers Eva-Maria

Johannes Gutenberg University of Mainz, Germany

Numerous studies report the association of miRNAs with extracellular vesicles (EVs). In most cases, EVs were harvested from cell culture-conditioned media containing fetal bovine serum (FBS) or a defined media supplement as nutrient. Recently, Wei et al. (2016, PMID: 27503761) reported that miRNAs are co-isolating with EVs when harvested in media supplemented with FBS or vesicle-depleted FBS. To avoid serum-associated miRNA contamination, we performed RNA-Seq of EV-associated miRNAs derived from primary oligodendrocytes cultured under serum-free conditions and subsequently validated miRNAs by RT-qPCR including media and supplement controls. Intriguingly, several EV-associated miRNAs were robustly detected in un-conditioned media subjected to the EV-isolation protocol and the media supplements NS21 and B27, which are routinely used for neural cell culture. RNAse and detergent treatment of NS21 removed most but not all of the contaminating miRNAs. Detailed analysis of NS21-supplement by screening individual components excluded BSA as major source of the miRNA contamination and identified a single component as carrier of miRNAs. Media supplement lacking the miRNA-carrying components however appears to affect cell viability, indicating that deprival of this specific supplement is not a solution to get rid of contaminating miRNAs. Based on these findings, we designed a new RNA-Seq strategy including EV-samples collected under conditions of supplementation or supplement-deprivation as well as supplement only control.

Taken together, our study shows that a single component of defined media supplements may carry major contaminating miRNAs into EV-samples. Consequently, analysis of EV-RNA needs refined strategies including respective media controls.

7:00pm - 11:59pmDINE: Event Dinner @ Bräustüberl
Date: Wednesday, 20/Mar/2019
9:00am - 10:30amMULTI1: Multi-Omics 1
Session Chair: Justin O'Grady, Quadram Insitute Bioscience, United Kingdom
HS 14 

The End of Medicine as we know it.

Harald H.H.W. Schmidt

Department of Pharmacology and Personalised Medicine, Faculty of Health, Medicine and Life Sciences, Maastricht University, The Netherlands

Following the IT revolution, the next socio-economic revolution appears to be a complete redefinition of health and disease, how we define them, how we handle them and how we finance this. Such revolutions follow upon a major crisis, and medicine is in a crisis. Existing drugs fail to provide benefit for most patients. The efficacy of drug discovery is in a constant decline and big pharma about to disappear in its current form by the end of the 2020s. Biomedical research has a poor translational success rate due to false incentives, lack of quality/reproducibility and publication bias. The most important reason and need for change, however, is our current concept of disease, i.e. mostly 19th/20th century-derived and based on organs or symptoms, but hardly every by mechanisms. Without a disease mechanism, however, no curative therapy is possible. Enabled by big-data and interdisciplinary research with applied bioinformaticians, the new Systems Medicine will lead to a mechanism-based redefinition of disease, precision diagnosis and therapy eliminating the need for drug discovery and a complete reorganization of how we teach, train and practice medicine.

Precision Medicine beyond Cancer: Why We Need New Multi-Omics Driven Definitions for Health & Disease

Thomas Wilckens

InnVentis Ltd, Germany

Building a Knowledge Network for Biomedical Research and a New Taxonomy of Disease” describes the concept to generate a molecularly-informed taxonomy of disease. This presentation addresses key challenges in data collection and labeling to achieve this goal.

Circulating miRNAs as Potential Biomarkers

Tim Kacprowski

Technical University of Munich, Germany

Non-cellular blood circulating microRNAs (plasma miRNAs) represent a promising source for the development of prognostic and diagnostic tools owing to their minimally invasive sampling, high stability, and simple quantification by standard techniques such as RT-qPCR. In this talk, I'll briefly present projects investigating the potential of plasma miRNAs both in a population-based cohort study and in patient cohorts for specific diseases.

We profiled circulating miRNAs in the population-based sohort study SHIP and investigated associations with age, sex, BMI. After regressing out technical parameters and adjusting for the respective other two phenotypes, 7, 15, and 35 plasma miRNAs were significantly (q < 0.05) associated with age, BMI, and sex, respectively. Adjustment for blood cell parameters slightly increased the number of age- or BMI-associated miRNAs but drastically reduced the number of sex-associted miRNAs. These findings emphasize that circulating miRNAs are strongly impacted by age, BMI, and sex. These parameters should be considered as covariates in association studies based on plasma miRNA levels. The established experimental and computational workflow can be used in future screening studies to determine associations of plasma miRNAs with defined disease phenotypes.

In a multicentre, prospective ACS cohort, 1002 out of 2168 patients presented with ST-segment elevation myocardial infarction (STEMI). Sixty-three STEMI patients experienced an adjudicated major cardiovascular event (MACE, defined as cardiac death or recurrent myocardial infarction) within 1 year of follow-up. From a miRNA profiling in a matched derivation case–control cohort, 14 miRNAs were selected for validation. Comparing 63 cases vs. 126 controls, miR-26b-5p levels (P=0.038) were decreased, whereas miR-320a (P=0.047) and miR-660-5p (P=0.01) levels were increased in MACE patients. MiR-26b-5p has been suggested to prevent adverse cardiomyocyte hypertrophy, whereas miR-320a promotes cardiomyocyte death and apoptosis, and miR-660-5p has been related to active platelet production. This suggests that miR-26b-5p, miR-320a, and miR660-5p may reflect alterations of different pathophysiological pathways involved in clinical outcome after ACS. These three miRNAs also discriminated cases from controls in age- and sex-adjusted Cox regression (AUC=0.718). Addition of the three miRNAs to both, the Global Registry of Acute Coronary Events (GRACE) score and a clinical model led to a net reclassification improvement of 0.20 in both cases.

9:00am - 10:30amMIQE&QC: MIQE Guidelines & Standardisation & Quality Control
Session Chair: Andreas Untergasser, Heidelberg University, Germany
HS 15 

Quality Standards in quantitative PCR; Specification, Validation, Controls and Standards

Peter Rossmanith

Vetmeduni Vienna, Austria

Introduction: The implementation of molecular methods such as real-time PCR for food pathogen detection is desired and reasonable. Nevertheless the obstacles of precise specification and meaningful validation are not yet overcome and therefore broad range use in food testing is not yet accomplished. Specification is generally based on the determination of the detection limit, the overall efficiency of the reaction and exclusivity and inclusiveness of the assay respectively. These parameters do not provide sufficient information about the real performance of the underlying enzymatic reaction. The validation according to ISO 16140, the validation of alternative methods, has many drawbacks based on its original sense and purpose, the comparison of microbiological methods. Evaluation of real-time PCR is therefore not significant with this process due to the different nature of molecular biological methods.

Purpose: Establishment of a significant specification and validation approach in consideration of the inherent qualities of real-time PCR.

Methods: A validation system including testing algorithms derived from software engineering; per se specification of the enzymatic reaction, controls covering all necessary steps and the investigation of surrounding parameters was designed. The whole approach is based on fundamental principles of systems theory and cybernetics. This alternative strategy includes every necessary detail thus leading to a maximum performance of the assay and most precise specification and validation of the whole analytical chain.

Results: We present the practical application of this new approach by example of an analytical chain for the detection of L. monocytogenes, including sample preparation, DNA isolation/purification and real-time PCR detection.

Significance: New approaches for the significant specification and validation of molecular biological methods are necessary to gain confidence in such methods and furthermore support widespread implementation. The whole system approach presented herein is an equivalent attempt, which effectively supports the standard validation method ISO 16140.

Directed Evolution of Enzymes for Streamlined and Reliable RT-qPCR and NGS Workflows

David Mark Schuster

Quantabio, United States of America

Reverse transcription remains an essential and sometimes problematic initial step in methods and workflows for the analysis of RNA by NGS or PCR-based amplification methods. Despite advancements in these technologies and the introduction of engineered reverse transcriptases, efficient conversion of RNAs that form stable secondary structures, and/or the presence of inhibitors in sample matrix can influence the efficacy of first-strand synthesis, introducing bias in RNA sequence coverage or transcript enumeration. This talk will describe novel thermostable, inhibitor tolerant RNA directed DNA polymerases obtained through our molecular screening and directed evolution program and their application to streamlined workflows for RT-PCR and NGS methods. Collectively, the properties of these new enzymes and associated reagent systems offer the promise to simplify, accelerate and improve the reliability and flexibility of detection and analysis of mRNA, noncoding RNA and viral targets.

Multiplex Mediator Probe Real-Time PCR: Optimisation and Guideline Development through Systematic Characterisation of Label Free Mediator Probes and Fluorogenic Universal Reporters

Michael Lehnert1,2, Elena Kipf1, Franziska Schlenker1, Roland Zengerle1,2, Nadine Borst1,2, Felix von Stetten1,2

1Hahn-Schickard, Freiburg, Germany; 2Laboratory for MEMS Applications, Department of Microsystems Engineering - IMTEK, University of Freiburg, Germany

Mediator Probe PCR is a powerful and robust real-time PCR technology for multiplex DNA detection and quantification. It uses label free mediator probes, for molecular detection of nucleic acids during DNA amplification, in combination with fluorogenic universal reporters for signal generation. During PCR, target sequence specific mediator probes are cleaved by the polymerase and a generic sequence, the mediator, is set free. In the second step the mediator binds to the universal reporter, where it is extended by the polymerase. This generates a strong fluorescence signal increase. Due to the separation of DNA detection and signal generation many advantages arise. Mediator probes are not limited in their design by properties of the target sequence and a standard set of highly optimised fluorogenic universal reporters can be used for multiplex Mediator Probe PCR, right from the start.1

In the last years Mediator Probe PCR evolved from an innovative new method to an optimised and robust multiplexing technology. This was achieved by systematic characterisation of its molecular processes, which again was advantaged by the separation of DNA detection and signal generation. A design of experiments (DoE) approach was used for the optimisation of Mediator Probes, focusing on their binding strengths.2 In parallel, a set of universal reporters with improved signal-to-noise ratios was established by successive testing over 40 molecular structures, with different fluorophore-quencher labels and configurations.1

As a result, distinct guidelines exist, which enable fast adaption of new DNA targets and facilitate multiplex Mediator Probe PCR design. The capability of the technology was shown by highly sensitive, precise and specific multiplex Mediator Probe real-time PCRs in different areas of molecular diagnostics. These fields include monitoring of oncological disease, detection of pathogens or analysis of food samples.1,3

1. Lehnert M, Kipf E, Schlenker F, Borst N, Zengerle R, von Stetten F. Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides. Anal. Methods. 2018;10:190. doi: 10.1039/C8AY00812D.

2. Wadle S, Lehnert M, Rubenwolf S, Zengerle R, von Stetten F. Real-time PCR probe optimization using design of experiments approach. Biomolecular detection and quantification. 2016;7:1–8. doi: 10.1016/j.bdq.2015.12.002.

3. Wadle S, Lehnert M, Schuler F et al. Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters. BioTechniques. 2016;61(3):123–8. doi: 10.2144/000114443.

11:00am - 12:30pmMULTI2: Multi-Omics 2
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
HS 14 

Systems Medicine - or - What I learned about Arnold Schwarzenegger while studying breast cancer survival.

Jan Baumbach

Technical University of Munich, Germany

On major obstacle in current medicine and drug development is inherent in the way we define and approach diseases. Here, we will discuss the diagnostic and prognostic value of (multi-)omics panels in general. We will have a closer look at breast cancer subtyping and treatment outcome, as case example, using gene expression panels - and we will discuss the current "best practice" in the light of critical statistical considerations. Afterwards, we will introduce computational approaches for network-based medicine. We will discuss novel developments in graph-based machine learning using examples ranging from Huntington's disease mechanisms via lung cancer drug target discovery back to where we started, i.e. breast cancer subtyping and treatment optimization - but now from a systems medicine point of view. We conclude that systems medicine and modern artificial intelligence open new avenues to shape future medicine.

Related paper: De novo pathway-based biomarker identification.

From Next Generation Sequencing to Next Generation Biomarkers: How Adaptive Focused Acoustics® is Transforming High-throughput Biology and Multi-omics Analyses

Nicolas Autret

Covaris, United Kingdom

“Standardization of sample preparation” is our core mission with a focus on clinical and pharmaceutical samples. As pre-analytical processes are increasingly recognized as the limiting factors for sensitivity and specificity of biomarker detection, this is especially relevant for highly advanced analytical methods such as Next Generation Sequencing or Mass Spectrometry. The AFA® (Adaptive Focused Acoustics®) process is isothermal and non-contact, providing precise process control, which is beneficial to a number of scientific disciplines in both advanced biological and chemical applications. Its high level of experimental condition control enables processes to be developed or improved upon very quickly, easily, and reproducibly. Covaris Focused-ultrasonicators may be programmed for intensity, duration, and duty factor, supporting a wide variety of applications, from gentle mixing to extreme compound reformatting and dissolution.

This talk will present some of the major applications driven by AFA (e.g. DNA and chromatin shearing, cfDNA isolation, nucleic acid and protein extraction from FFPE). Many of these were launched recently, including a series of kits in the truChIP/truXTRAC product line. We will also discuss insights into current developments in automation and robotization, introducing the first focused-ultrasonicator integrated on a liquid handler deck with precise energy, control, and a proprietary scanning process. This instrument provides increased workflow efficiency, full automation, and high-throughput sample prep workflows.

Finding Signatures, Fingerprints and Prognostic Biomarkers in Large Biomedical Datasets: Computational and visual Approaches.

Andrej-Nikolai Spiess

UKE Hamburg, Germany

In the last ten years, the amount of experimental data acquired by high-throughput technologies such as microarrays and RNA sequencing (RNAseq) has increased exponentially and resulted in partly Gigabyte-sized expression matrices. It is not uncommon that the researcher is faced with tables of 20000 rows (transcripts, genes) and 2000 columns (samples), necessitating mathematical, computational and visual approaches that are specifically tailored to these high-dimensional datasets. Frequently, the wet lab scientist “outsources” these analyses to an associated bioinformatics department, getting in return an often black box-type sophisticated analysis on which to rely. Here, it is important that a common ground on existing analysis approaches of this kind of data must be established.

In my talk, I will give a concise and comprehensive overview on existing methods to analyze large-scale gene expression data. Without going into deep mathematical details – these can be obtained from the literature – I will provide an outline on the important aspects and idiosyncrasies of current methodology based largely on the 2D- and 3D-visual depiction of data. Starting from very basic topics such as data cleaning/normalization/scaling, I will emphasize on efforts to uncover the intrinsic signature of the data (without imposing any presumptions), based on unsupervised clustering methods such as hierarchical clustering and dimension reduction methods such as PCA (linear) or the recent t-SNE approach (non-linear). I will demonstrate that in published datasets, the intrinsic structure of the data can be significantly different to the one assumed or defined by the experimental setup (such as batch effects). Next, I will give a summary on how to filter signatures that discriminate between different cellular states and how to use computationally expensive methods (bootstrapping, cross-validation) to avoid extracting signatures that perform great on the training set but bad on independent data (overfitting). Along these lines, a short introduction on recent machine learning approaches such a random forests, neural networks and gradient boosting will be delivered, and their advantage in finding predictive biomarkers and reduced discriminator sets through feature selection. For all the discussed approaches, I will also highlight the different pitfalls, for instance when to correct for multiple testing, why to never perform a statistical test before clustering, and (quite crucially) the identification of differential expression that is mimicked by the shifting of cellular proportions.

11:00am - 12:30pmqPCR-DA1: qPCR Data Analysis 1
Session Chair: Stefan Rödiger, Brandenburg University of Technology Cottbus - Senftenberg, Germany
HS 15 

GeneGini: Assessment via the Gini Coefficient of Reference "Housekeeping" Genes and Diverse Human Transporter Expression Profiles.

Philip Day1, Stephen O'Hagan1, Marina Wright Muelas1, Emma Lundberg2, Douglas Kell1

1University of Manchester, United Kingdom; 2KTH Royal Institue of Technology, Stockholm, Sweden

The expression levels of SLC or ABC membrane transporter transcripts typically differ 100- to 10,000-fold between different tissues. The Gini coefficient characterizes such inequalities and here is used to describe the distribution of the expression of each transporter among different human tissues and cell lines. Many transporters exhibit extremely high Gini coefficients even for common substrates, indicating considerable specialization consistent with divergent evolution. The expression profiles of SLC transporters in different cell lines behave similarly, although Gini coefficients for ABC transporters tend to be larger in cell lines than in tissues, implying selection. Transporter genes are significantly more heterogeneously expressed than the members of most non-transporter gene classes. Transcripts with the stablest expression have a low Gini index and often differ significantly from the "housekeeping" genes commonly used for normalization in transcriptomics/qPCR studies. PCBP1 has a low Gini coefficient, is reasonably expressed, and is an excellent novel reference gene. The approach, referred to as GeneGini, provides rapid and simple characterization of expression-profile distributions and general improved normalization of genome-wide expression-profiling data will be described

GenEx – The Ultimate Software for Analysis of Transcriptomic Data.

Amin Forootan1, Björn Sjögreen1, Mikael Kubista2

1Multid Analyses AB, Sweden; 2TATAA Biocenter, Sweden

With the emergence of RNA sequencing (RNASeq) transcriptome profiling entered a new era. High throughput high quality whole transcriptome data can today be collected routinely. The challenge is no longer acquiring data but rather analyzing and interpreting them. Analysis includes validating data quality, merging runs, normalizing the data, comparing experimental conditions, testing hypothesis and interpreting the results. GenEx is the most used software for qPCR data analysis and with the launch of GenEx 7, here at the 9th Gene Quantification Event, also RNASeq data can readily be analyzed. GenEx is developed for experimentalists, with a user-friendly intuitive interface that provides a smooth analytical workflow for statistical analyses of the data in compliance with guidelines when relevant. Very large data sets, typical of RNASeq, are easily and rapidly handled and graphical interfaces allow interactive analyses with powerful methods such as DESeq2, and Normfinder for normalization, t-test, Mann-Whitney, Wilcoxon’s test and ANOVA models for group comparisons, hierarchical clustering, self-organizing maps (SOM) and principal component analysis (PCA) for clustering, dynamic PCA with statistical filters for variable selection to find the most relevant expression markers, kinetic PCA for time studies, survival analysis to compare treatments, and artificial neural network (ANN) and support vector machines (SVM) to build predictive models. GenEx 7 is continuously updated to include new methods and strategies as they become available and to maintain compatibility with qPCR and NGS instrument software, computer operating systems, and graphical and printer routines. GenEx 7 is the only data analysis software supported by the majority of leading instrument and solution providers.

Why reporting Cq or delta-Cq is senseless.

Jan M Ruijter

Academic Medical Center, Amsterdam, the Netherlands, Netherlands, The

With the introduction of quantitative PCR (qPCR) it was assumed that the amplification efficiency, the fold-increase per cycle, was always close to 2. This simplification allowed the use of the so-called comparative Cq equation to calculate the fold-difference between target and reference genes in treated and control tissues. Over the years the original equation (2-ΔΔCq) seems to have lost its base and the minus sign. The remainder became so ingrained in qPCR-based papers that ‘ddCq’ currently seems to be the unit in which qPCR data are measured and have to be reported. However, the variations in annotation of the figure axes make that the presented data often cannot be interpreted.

The Cq value is defined by the general principle that the position of the amplification curve with respect to the cycle-axis, reflected in the Cq value, is a measure for the initial target quantity: the ‘later’ the curve, the higher the Cq value and the lower the starting quantity of the target-of-interest. However, this position is also dependent on the amplification efficiency. Therefore, reporting only ddCq implicitly accepts unvalidated assumptions about the amplification efficiencies involved. Reported Cq values can only be interpreted with the simplifying, and false, assumption that every PCR assay in the experiment is 100% efficient. Because of this assumption, the interpretation of Cq values always leads to an unknown bias.

The bias that is introduced by ignoring the actual PCR efficiency of target and reference genes can be prevented with the calculation of the so-called efficiency-corrected target quantities or fold-differences. This was already proposed in the early years of this millennium and is recommended in the MIQE guidelines. Indeed, such efficiency-corrected target quantities are reported by a number of qPCR data analysis methods published over a decennium ago. However, this need for efficiency-correction of qPCR results is still largely ignored by researchers, reviewers and publishers. This common shortcoming of the PCR research community may be the main reason for the limited reproducibility of reported qPCR results.

12:30pm - 2:00pmPO3: Wednesday Lunch Poster Session
All posters will be displayed in parallel at all three poster sessions PO1, PO2 & PO3 => the poster viewing can be done from "Monday Evening Main Poster Session" till "Wednesday Lunch Poster Session"
Lower Level 
12:30pm - 2:00pmLU3: Lunch Wednesday
2:00pm - 4:00pmAMDx2: Advanced Molecular Diagnostics 2
Session Chair: John Mackay, dnature Diagnostics & Research Ltd, New Zealand
HS 14 

“Saving The Bees Is Burning Down The House” - Triplex qPCR Using Dual-Target Assays For The Highly Pathogenic Bacteria American Foulbrood, Using Novel eDNA Methods.

John Mackay, Tammy Waters

dnature diagnostics & research Ltd, Gisborne, New Zealand

American Foulbrood (AFB) is the most devastating pathogen of honeybee diseases. It is estimated that AFB has a minimum direct cost of more than US$7 million dollars per year to New Zealand beekeepers. Worse, the incidence of the disease is increasing at an estimated 15% per year. We have developed a multiplex qPCR for AFB and are using this to screen bee and honey samples, as well as testing new sampling strategies to predict the development of this disease and prevent further spread.
A snapback high resolution melting method for multiple SNP resolution in apiculture will be also be described.

A Portable, Accurate, and Cost-Effective Strategy for Rapid On-Site Authentication and Characterization of Commercially Important Species and Pathogens Using Bio Molecular Systems’ MIC qPCR Cycler

Jesus Aquiles Sanchez, Lawrence Wangh

Thermagenix, Inc, Natick, Massachusetts, United States of America

PROBLEM: In the seafood industry, mislabeled products disguising lesser-value/lower-quality species unfairly compete for profits, harm brands/consumer trust, and prevent proper safety tests for species-specific hazards and pathogens. In agriculture, invasive/destructive species continuously threaten crops and farmers’ livelihoods. Routine large-scale species testing imperative for these and many other industries is currently not possible due to the high-cost and complexities of species DNA sequencing and the complications of using a different DNA test for each species. SOLUTION: In response, ThermaGenix developed a broadly-applicable strategy for rapid and cost-effective identification of up to hundreds of species and pathogens in a single-tube test. One set of reagents in the test identifies multiple species individually without sequencing. ThermaGenix’s universal species DNA tests run on the MIC qPCR Cycler, a highly accurate, portable, and affordable instrument from Bio Molecular Systems for field applications. TECHNOLOGY: ThermaGenix’s High Precision PCR (based on ThermaStop™, a proprietary reagent for error-free DNA amplification) coupled with paired sets of positive/negative Nielsen hybridization probes convert any species-specific DNA sequences into highly accurate fluorescent signatures. Sequence-specific fluorescent signatures are then automatically compared against a reference library for immediate species identification. APPLICATIONS: FASTFISH-ID™, ThermaGenix’s first product for the MIC qPCR Cycler, provides rapid on-site DNA authentication of >700 individual species in commercial fish products in a single-tube test in about two hours. Another ThermaGenix test identifies any of 17 bacterial and fungi pathogens associated with sepsis in a single-tube. SIGNIFICANCE: ThermaGenix’sHigh-Precision PCR reagents and platform technology together with the high accuracy, reproducibility, and portability of Bio Molecular Systems’MIC qPCR Cycler provides an innovative turnkey solution for rapid on-site identification of large numbers of species and pathogens in a single-tube using a single set of reagents. Application target areas include on-site food integrity and safety testing, detection of invasive pest species and their pathogens, environmental bioassessment programs; additional uses include point-of-care diagnostics for cancer, infectious diseases, and other fields.

Development Of An Event-Specific qPCR Method For Detection Of Genetically Modified Alfalfa

Patrick Guertler1, Lutz Grohmann2, Heike Naumann3, Melanie Pavlovic1, Ulrich Busch1

1Bavarian Health and Food Safety Authority, Germany; 2Federal Office of Consumer Protection and Food Safety, Germany; 3Lower Saxony State Office for Consumer Protection and Food Safety, Germany

Genetically modified (gm) plants (GMP) have gained importance since commercialization in 1996. Cultivation areas increased from 1.7 million hectares in 1996 to almost 190 million hectares in 2017. In Europe, GMPs need to be authorized before being placed on the market and food and feed products containing authorized GMPs need to be labeled above a gm content of 0.9 %. Non-authorized products must not be placed on the EU market.

One of the emerging GMP species is alfalfa (Medicago sativa), which is one of the most important forage crops worldwide. Modified gm alfalfa events J101 and J163 gained herbicide tolerance against glyphosate by incorporating a CTP2-CP4 epsps gene. In event KK179, the RNA interference technique was used to knock-out the caffeoyl-CoA-3-O-methyltransferase (CCOMT) translation. CCOMT is a key enzyme in the lignin pathway and a knock-out leads to an improved digestibility for ruminants. Gm alfalfa is commercially cultivated in the US and in Canada.

In order to develop a qPCR-based detection method, we designed plasmids for each gm alfalfa event, based on published patent sequences. Further, we designed primers and a hydrolysis probe targeting the junction sequence spanning the plant genome and the transgenic insert (=event-specific detection). Plasmids were quantified using ddPCR and used for optimization and in-house validation of the methods. An estimated LOD95% of 10 copies per PCR was observed and PCR efficiencies of 95 – 97 % were achieved. Different qPCR instruments and PCR conditions were applied to test for robustness. Certified reference material for different GMP was used to test for specificity. No unspecific amplification signal was observed for any of the developed methods.

An inter-laboratory comparison study with seven participating laboratories was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters. Our cooperation partner (Federal Office of Consumer Protection and Food Safety, Berlin) was able to procure ground seed material for all three gm alfalfa events, which could be used in this inter-laboratory comparison study. All participants reported qPCR efficiencies between 95.9 % and 106.9 % and all laboratories were able to detect 10 nominal copies in twelve replicates. All results underline the suitability of the methods for the specific detection of gm alfalfa events J101, J163 and KK179.

A full collaborative trial validation study of the developed methods is planned for 2019.

MyPOLS Biotec: Shaping DNA Polymerases For Your Needs

Moritz Welter1,2, Andreas Marx2, Ramon Kranaster1

1myPOLS Biotec GmbH, Technologiezentrum Konstanz, Blarerstraße 56, 78462 Konstanz, Germany; 2Chair of Organic Chemistry / Cellular Chemistry, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany

Based on their experience in basic and applied research on DNA polymerases, Dr. Ramon Kranaster and Prof. Dr. Andreas Marx founded myPOLS Biotec GmbH in Konstanz, Germany, as a spin-off from the University of Konstanz in 2014. Since then, myPOLS Biotec’ business activities, focused on the development of innovative applications of DNA polymerases, turned out to be very successful.

Off-the shelf, myPOLS Biotec offers DNA polymerase-based products like DIRECT BLOOD GENOTYPING KITS ­ that tolerate blood ingredients in real-time PCR allowing genotyping directly from blood specimen, thereby saving time and money by omitting the nucleic acid extraction step; HiDi DNA POLYMERASE – a DNA polymerase that provides significantly enhanced selectivity of matched versus mismatched primers during PCR extension steps, rendering it the first choice in mutation-detection assays via allele-specific PCRs; VOLCANO2G DNA POLYMERASE – an enzyme that is capable of performing reverse transcription PCR without the need of an isothermal reverse transcription step to promote “zero-step” RT-PCR; Kits for DIRECT PCR FROM PLASMA – are currently developed in collaboration with the University of Konstanz that will allow analysis directly from blood plasma e.g., for the detection of cancer mutations by real-time, liquid biopsy PCRs.

To provide solutions for customized in-vitro-diagnostics, myPOLS Biotec develops and produces tailored products for IVD applications. For instance, the PCR LYOBEADS and LYOCAKES product lines: As freeze-dried ready to use master mixes, they can be shipped and stored at room-temperature and contain all components necessary (i.e., enzymes, primers, and probes) for a rapid, sensitive and reproducible detection and quantification of nucleic acid targets.

In contract research projects, myPOLS Biotec offers its specialized knowledge as a highly reliable, and transparent partner in challenging research projects based on DNA polymerases and their tailoring for advanced applications.

In the presentation, Ramon Kranaster will introduce the company myPOLS Biotec and provide an overview about newest developments, applications and products.

2:00pm - 4:00pmqPCR-DA2: qPCR Data Analysis 2
Session Chair: Jan M Ruijter, Academic Medical Center, Amsterdam, Netherlands, The
HS 15 

Integration of DNA Melting Curve Analysis In qPCR Data Analysis

Maurice J.B. van den Hoff1, Quinn D. Gunst1, Adrian Ruiz-Villalba2, Carl Wittwer3, Jan M. Ruijter1

1Amsterdam UMC, location AMC, Depart. Medical Biology, Amsterdam, The Netherlands; 2Foundation of Applied Medical Research, University of Navarra, Pamplona, Spain; 3University of Utah Health Sciences Center, Department of Pathology, Salt Lake City, UT, USA

Quantitative PCR (qPCR) allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, analysis of the results of 101 validated SybrGreen I-based assays for genes related to the Wnt-pathway in 5 different cardiac compartments frequently showed the amplification of nonspecific products, most probably primer-dimers. A detailed survey of these data revealed that the occurrence of nonspecific products is not related to Cq value or the PCR efficiency. qPCRs amplifying both specific and non-specific products can easily be identified when a melting curve analysis is performed. Currently, qPCRs that amplify both the specific and (a) nonspecific product(s) need to be excluded from further analysis because the quantification result is meaningless.

A model was developed, allowing the quantification of a qPCR in which the correct product together with additional off-target products is amplified. This model is based on the analysis of the melting peaks and the assignment of the total fluorescence at the end of the reaction to either the correct product or to other products. The fraction of fluorescence due to the amplification of the correct product can then be used to correct the quantification result (Cq value or target quantity, N0) that was derived from the observed amplification curve.

This correction method, and a program to analyze melting curves, was tested for the 101 different validated qPCR assays in different biological tissues and for model experiments with known concentrations of different products. The results of these tests show improvement of the sensitivity of SybrGreen I-based assays and avoid erroneous conclusion.

Fundamentals for the Automatic Classification of Quantitative PCR AmplificationCurves - A Biostatistical Approach

Stefan Rödiger1, Andrej-Nikolai Spiess2, Michał Burdukiewicz3

1Brandenburg University of Technology Cottbus - Senftenberg, Germany; 2University Medical Center Hamburg-Eppendorf, Germany; 3Warsaw University of Technology, Poland

Quantitative polymerase chain reaction (qPCR) is a widely used bioanalytical method in forensics, human diagnostics and life sciences. With this method nucleic acids are detected and quantified. In qPCRs, the enzymatic amplification of the target DNA (amplicon) is monitored in real-time by fluorescent reporter molecules marking the synthesized PCR products cycle by cycle. The measured fluorescence is proportional to the amplicon amount.
For diagnostic and forensic applications in particular, the question arises for example as to whether an amplification reaction is negative or positive. Of interest is also and automatic classification of the quality of amplification curves. Until now, such classification was usually performed manually or on the basis of fixed threshold values. However, this approach is error-prone if inadequate thresholds are used or the user performs the classification subjectively based on his experience.
Therefore, the classifications of the same sample may not be identical for different users. Such errors are problematic because they can lead to an erroneous judgement. Therefore we developmed a scientific open source software, called PCRedux ( With this software, predictors (features) of amplification curves can be calculated automatically. A predictor is a quantifiable informative property of an amplification curve. A set of statistical algorithms for the calculation of predictors os proposed. The work also shows how predictors can be used in tests and logical combinations to perform machine-based classifications.
All scientific work depends on the data, with open data in particular being regarded as a cornerstone of science. Since no data sets of classified amplification curves were available, the work also deals with the aggregation, management and distribution of classified qPCR data sets. Manual classification of amplification curves is time-consuming and error-prone, especially for large data sets. To improve this, auxiliary tools have been developed.
A open approach for curve-shape based group classification was proposed.

GEAR: The Genome Analysis Server Eases Wet-Lab Data Analysis

Tobias Rausch1, Markus Hsi-Yang Fritz2, Vladimir Benes1, Andreas Untergasser1,3

1European Molecular Biology Laboratory, Genomics Core Facility, Heidelberg, Germany; 2European Molecular Biology Laboratory, Genome Biology Unit, EMBL, Heidelberg, Germany; 3Heidelberg University, Germany

The genome analysis server (GEAR: is a wide collection of tools supporting molecular biologists in everyday lab tasks. An enhanced version of Primer3Plus allows the selection of primers for many use cases like detection, qPCR, cloning and sequencing. Secondary structures are now also drawn and can be evaluated by the researcher. Silica can perform in-silico PCRs on a selected genome with a set of provided primers. It localizes primer binding sites and calculates the amplicons. The Wily-DNA-Editor is a DNA sequence editor supporting genbank files and sufficient for common plasmid manipulation tasks. Users can edit or reverse complement the sequence, find restriction sites, draw restriction maps, calculate digests, find open reading frames, translate sequences and allows a custom feature annotation. Due to its JavaScript nature all data are processed in the user's browser without being transferred to the server. Teal, Sage and Indigo display Sanger trace files and extract the sequence information. They ease the evaluation by aligning the trace file to a genome or a provided reference sequence highlighting the found differences. Last, the RDML tools support users in the evaluation and the padding of RDML files. The user can validate the files against the schema format description, fix common errors and build RDML files from table data. Ultimately, the RDML tools will allow to edit and analyze RDML files as well as evaluating compliance with MIQE.

These tools are very useful for molecular biologists as they solve common lab tasks and enable to work at any computer with internet connection and a current browser - without the need of installing software locally. The code is open source and users that due to legal restrictions cannot send their data on servers over the internet may opt to install an own version of gear on a local server and process their data in house.

Digital PCR provides new challenges. The RDML format has to be extended to support dPCR data in an efficient way and the tools have to be extended to visualize the data. Last, we would like to draw attention to a session on RDML and digital PCR were everybody is invited to provide suggestions on the further development of RDML.

DAILYqpcr – An Application For Revolutionizing Designing, Storing, And Analyzing QPCR Experiments

Stephan Pabinger, Anna Majewski, Manuela Hofner, Walter Pulverer, Priska Bauerstätter, Stefanie Eile, Julie Krainer, Andreas Weinhäusel, Klemens Vierlinger

AIT - Austrian Institute of Technology, Austria

Quantitative real-time polymerase chain reaction (qPCR) is a standard method in most laboratories for quantification of gene expression. However, the streamlined design of experiments, its analysis, and the controlled storage of results is still an unresolved problem.

Here we present a novel tool that allows the seamless integration between lab and data analysis workflows with a strong focus on usability. DAILYqpcr is a Python and R based web-application that is centered around two main aspects: (i) an interactive designer to outline the qPCR experiment before it is processed in the laboratory; (ii) a collection of analysis workflows tailored to specific use-cases such as methylation analysis or differential gene expression.

Instead of offering a plethora of methods and tools where the user needs to know exactly how to use them, we focus on providing wizard-like analysis solutions for specific use-cases customized to the tasks and needs of the scientists. Depending on the type of experiment, the appropriate analysis tools and parameters are selected and configured for the user. This allows a streamlined experience reducing the analysis time while at the same time avoiding the misuse of methods.

As an example, the workflow assay validation starts with reading in the data from the thermocycler (currently Lightcycler and Fluidigm are supported), continues with customized quality assessment steps, and outputs performance characteristics and interactive plots about each tested assay. Throughout the workflow the user is guided through the necessary steps, each of which is stored to allow resuming the analysis at a later timepoint.

The integrated database stores data, settings and results, hence allowing researchers to search for analysis outcomes, samples, assays, designs and other resources. For example, users can check whether an assay has already been applied for a specific set of genes or if samples were already used in other experiments. Furthermore, the application incorporates widely used R-packages, provides convenient import and export mechanisms, and can be easily extended with new use-cases.

In summary, we present a novel tool that streamlines the experience of working with qPCR data and provides a novel way to design and analyze qPCR experiments.

4:00pm - 4:30pmFW: Farewell Session
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
HS 14 
Date: Thursday, 21/Mar/2019
9:00am - 5:00pmWS1: Workshop Day 1
Workshop Rooms 
Date: Friday, 22/Mar/2019
9:00am - 5:00pmWS2: Workshop Day 2
Workshop Rooms