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).
Location: HS 15 |
ZHG -- Lecture Hall 15
|Date: Monday, 18/Mar/2019|
|2:00pm - 6:00pm||NGS-DA: NGS & NGS Data Analysis|
Session Chair: Jo Vandesompele, Ghent University & Biogazelle, Belgium
Session Chair: David Langenberger, ecSeq Bioinformatics GmbH, Germany
RNA Atlas: A Nucleotide Resolution Map Of The Human Transcriptome
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
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
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
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
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.
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
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.
|Date: Tuesday, 19/Mar/2019|
|8:30am - 12:30pm||LB: Liquid Biopsy|
Session Chair: Jo Vandesompele, Ghent University & Biogazelle, Belgium
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
Cell-Free Tumor DNA Analysis In Liquid Biopsy Using Ultrasensitive Sequencing
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
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
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.
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
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
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
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 - 2:00pm||Journal: BDQ Editors Meeting|
Session Chair: Jim Huggett, University of Surrey, United Kingdom
Session Chair: Justin O'Grady, Quadram Insitute Bioscience, United Kingdom
|2:00pm - 6:00pm||ISEV: 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
Mesenchymal Stem Cell-derived Extracellular Vesicles: a Potential New Tool in Regenerative Medicine
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
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
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
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
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
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
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.
|Date: Wednesday, 20/Mar/2019|
|9:00am - 10:30am||MIQE&QC: MIQE Guidelines & Standardisation & Quality Control|
Session Chair: Andreas Untergasser, Heidelberg University, Germany
Quality Standards in quantitative PCR; Specification, Validation, Controls and Standards
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
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
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 - 12:30pm||qPCR-DA1: qPCR Data Analysis 1|
Session Chair: Stefan Rödiger, Brandenburg University of Technology Cottbus - Senftenberg, Germany
GeneGini: Assessment via the Gini Coefficient of Reference "Housekeeping" Genes and Diverse Human Transporter Expression Profiles.
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.
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.
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.
|2:00pm - 4:00pm||qPCR-DA2: qPCR Data Analysis 2|
Session Chair: Jan M Ruijter, Academic Medical Center, Amsterdam, Netherlands, The
Integration of DNA Melting Curve Analysis In qPCR Data Analysis
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
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.
GEAR: The Genome Analysis Server Eases Wet-Lab Data Analysis
1European Molecular Biology Laboratory, Genomics Core Facility, Heidelberg, Germany; 2European Molecular Biology Laboratory, Genome Biology Unit, EMBL, Heidelberg, Germany; 3Heidelberg University, Germany
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
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.