Conference Agenda

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

 
Session Overview
Date: Wednesday, 20/Mar/2019
9:00am - 10:30amMULTI1: Multi-Omics 1
Session Chair: Justin O'Grady, Quadram Insitute Bioscience, United Kingdom
HS 14 
 

The End of Medicine as we know it.

Harald H.H.W. Schmidt

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

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



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

Thomas Wilckens

InnVentis Ltd, Germany

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



Circulating miRNAs as Potential Biomarkers

Tim Kacprowski

Technical University of Munich, Germany

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

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

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

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

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

Peter Rossmanith

Vetmeduni Vienna, Austria

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

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

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

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

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



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

David Mark Schuster

Quantabio, United States of America

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



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

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

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

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

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

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

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

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

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

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

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

Jan Baumbach

Technical University of Munich, Germany

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

Related paper: De novo pathway-based biomarker identification.

https://www.ncbi.nlm.nih.gov/pubmed/28934488



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

Nicolas Autret

Covaris, United Kingdom

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

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



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

Andrej-Nikolai Spiess

UKE Hamburg, Germany

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

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

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

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

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

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

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



GenEx – The Ultimate Software for Analysis of Transcriptomic Data.

Amin Forootan1, Björn Sjögreen1, Mikael Kubista2

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

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



Why reporting Cq or delta-Cq is senseless.

Jan M Ruijter

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

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

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

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

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

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

John Mackay, Tammy Waters

dnature diagnostics & research Ltd, Gisborne, New Zealand

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



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

Jesus Aquiles Sanchez, Lawrence Wangh

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

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



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

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

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

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

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

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

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

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



MyPOLS Biotec: Shaping DNA Polymerases For Your Needs

Moritz Welter1,2, Andreas Marx2, Ramon Kranaster1

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

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

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

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

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

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

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

Integration of DNA Melting Curve Analysis In qPCR Data Analysis

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

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

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

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

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



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

Stefan Rödiger1, Andrej-Nikolai Spiess2, Michał Burdukiewicz3

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

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



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

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

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

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

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

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



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

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

AIT - Austrian Institute of Technology, Austria

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

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

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

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

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

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

 
4:00pm - 4:30pmFW: Farewell Session
Session Chair: Michael W Pfaffl, Technical University of Munich, Germany
HS 14