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Location: HS 14 |
ZHG -- Lecture Hall 14
|Date: Monday, 18/Mar/2019|
|10:00am - 10:30am||WE: Welcome Session|
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
Session Chair: Stephen Andrew Bustin, Anglia Ruskin University, United Kingdom
|10:30am - 12:30pm||NGB: Next Generation Biomarkers|
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
Towards a Universal Diagnostic Test for Infection Using Rapid Nanopore Metagenomics
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?
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
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
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:
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.
|2:00pm - 6:00pm||SC&MG: Single-Cell & Micro-Genomics|
Session Chair: Mikael Kubista, TATAA Biocenter AB, Sweden
Session Chair: David Ruff, Mission Bio, Inc., United States of America
Spatial Transcriptomics - Bridging Histology and RNA Sequencing
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
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
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.
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?
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
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
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.
|Date: Tuesday, 19/Mar/2019|
|8:30am - 12:30pm||dPCR: digital PCR|
Session Chair: Jim Huggett, University of Surrey, United Kingdom
Session Chair: Afif Abdel Nour, Universite Saint Esprit de Kaslik, Lebanon (Lebanese Republic)
Biometrology, What Is It And What Could It Mean For Biomolecular Research?
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
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
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!
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
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
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.
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.
|2:00pm - 6:00pm||AMDx1: Advanced Molecular Diagnostics 1|
Session Chair: Carl T Wittwer, University of Utah, United States of America
Session Chair: Mikael Kubista, TATAA Biocenter AB, Sweden
Rapid qPCR and the Future of Molecular Diagnostics
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
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
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.
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
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
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
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.
|Date: Wednesday, 20/Mar/2019|
|9:00am - 10:30am||MULTI1: Multi-Omics 1|
Session Chair: Justin O'Grady, Quadram Insitute Bioscience, United Kingdom
The End of Medicine as we know it.
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
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
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.
|11:00am - 12:30pm||MULTI2: Multi-Omics 2|
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
Systems Medicine - or - What I learned about Arnold Schwarzenegger while studying breast cancer survival.
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
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.
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.
|2:00pm - 4:00pm||AMDx2: Advanced Molecular Diagnostics 2|
Session Chair: John Mackay, dnature Diagnostics & Research Ltd, New Zealand
“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.
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 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
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
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
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.
|4:00pm - 4:30pm||FW: Farewell Session|
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany