High resolution NGS-based HLA-typing using in-solution targeted enrichment

Michael Wittig1, Jarl Andreas Anmarkrud2,3,4, Jan Christian Kässens5, Simon Koch6, Michael Forster1, Eva Ellinghaus1, Johannes E.R. Hov2,4,7, Sascha Sauer8, Manfred Schimmler5, Malte Ziemann9, Siegfried Görg9, Frank Jacob6, Tom Hemming Karlsen2,4,7, Andre Franke1
1Christian-Albrechts-University Kiel, Institute of Clinical Molecular Biology, Kiel, Germany; 
2Norwegian PSC Research Center, Department of Transplantation Medicine, Divison of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway; 
3K.G. Jebsen Inflammation Research Center, Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 
4Research Institute of Internal Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Oslo, Norway; 
5Christian-Albrechts-University of Kiel, Department of Computer Science, Kiel, Germany; 
6Muthesius Academy of Fine Arts and Design, Kiel, Germany;
7Section of Gastroenterology, Department of Transplantation Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway; 
8Max-Planck Institute for Molecular Genetics, Berlin; 
9University of Lübeck, Institute of Transfusion Medicine, Lübeck, Germany

Abstract
The human leukocyte antigen (HLA) complex contains the most polymorphic genes in the human genome. The classical HLA class I and II genes define the specificity of adaptive immune responses. Genetic variation at the HLA genes is associated with susceptibility to autoimmune and infectious diseases and plays a major role in organ transplantation and immunology. Currently, the HLA genes are characterized using Sanger- or next-generation sequencing (NGS) of a limited amplicon repertoire or labeled oligonucleotides for allele-specific sequences. We developed a highly automated HLA typing method for NGS. The method employs in-solution targeted capturing of the classical class I (HLA-A, HLA-B, HLA-C) and class II HLA genes (HLA-DRB1, HLADQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1) followed by an amplification of the captured DNAs. The method was tested on 357 commercially available DNA samples with known HLA alleles obtained by classical typing. Our results showed on average an accurate allele call rate of 0.99 in a fully automated manner, identifying also errors in the reference data. Finally, the method provides the flexibility to add further enrichment target regions.

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Sample to Insight – analysis, visualization and interpretation of next generation sequencing and gene expression data

Anne Arens
QIAGEN GmbH, Germany

Abstract
QIAGEN provides complete sample to insight solutions for your next-generation sequencing (NGS) workflow including bioinformatics analysis. High-quality, target-enriched samples and streamlined library preparation procedures can be combined with easy-to-use, graphical user-interface-guided data analysis tools. Our solutions deliver focused and accurate results and allow you to overcome the bottlenecks in your NGS data analysis and interpretation by using our manually curated knowledgebase.
Accepting data from any major sequencing platform, CLC Genomics Workbench and CLC Cancer Research Workbench align sequencing reads to the reference genome, and call germline or somatic variants for targeted, whole exome, transcriptome and genome sequencing. Due to seamless integration of our software solutions, variants can be uploaded directly from CLC Cancer Research Workbench to Ingenuity Variant Analysis for fast and accurate filtering, annotation, and interpretation. Ingenuity Variant Analysis combines analytical tools and integrated content to help you rapidly identify and prioritize variants by drilling down to a small, targeted subset of compelling variants based both upon published biological evidence and your own knowledge of disease biology.
For gene expression analysis, CLC Genomics Workbench and CLC Cancer Research Workbench provide tools for RNA-Seq as well as expression chip support. Streamlined integration with Ingenuity Pathway Analysis allows for direct upload, analysis, and interpretation of your expression data. Ingenuity Pathway Analysis provides a rapid assessment of the signaling and metabolic pathways, biological processes, and upstream regulators that are most significantly perturbed in a dataset of interest. Ingenuity Pathway Analysis can also be used for expression data generated with other experimental platforms, including qPCR or microRNA.
Ingenuity Variant Analysis and Ingenuity Pathway Analysis leverage our unique repository of known human genes (as well as mouse and rat genes), proteins, phenotypes, variants, pathways, pathway regulators, drugs, and other compounds as well as their interactions. The Ingenuity knowledgebase is mostly composed of several millions of up-to-date experimental findings extracted from peer-reviewed journals that have been curated by MD/PhD level scientists, and third party information from public high quality databases. Recently our variant related records have been augmented by the variants reported in BIOBASE’s HGMD professional database.
We will illustrate the capability of our software solutions by introducing use cases related to DNA-Seq and RNA-Seq.

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Inferring Ribosome Dynamics From mRNA Degradome Sequencing

Vicent Pelechano1, Wu Wei2,3, Lars M Steinmetz1,2,3
1European Molecular Biology Laboratories, Genome Biology Unit, Germany; 
2Stanford Genome Technology Center, Stanford University, USA; 
3Department of Genetics, Stanford University School of Medicine

Abstract
We have developed an optimized protocol (5PSeq) including unique molecular identifiers that allows measuring all 5’Phosphate mRNA degradation intermediates present in a sample. mRNA degradation intermediates have been previously analysed to identify RNA endonucleolitic cleavage sites for 5’-3’ RNA degradation mutants both in plants and yeast. However, what are the specific factors contributing to the presence of 5’P sites and how do their abundance vary across the genome and physiological conditions has not been studied.
We have investigated the 5’ positions of degradation mRNAs with 5PSeq in multiple physiological conditions and drug treatments in S. cerevisiae. Our genome-wide analysis shows that ribosomes act as a general barrier for mRNA degradation, suggesting that the previously proposed mRNA co-translation degradation is not only possible, but also pervasive in the genome. Analysis in other organism shows that this process is evolutionary conserved. By comparing 5PSeq with Ribosome Profiling, we show that the detailed study of the mRNA degradation intermediates produced by the endogenous RNA degradation machinery allows reconstructing ribosome dynamics in vivo. 5PSeq is a straightforward method that does not require any polyribosome fractionation or RNAse foot-printing and that can be easily applied to any previously purified RNA. Additionally this drug-free approach can be used to study ribosome pausing sites that in alternative methods such as Ribosomal Profiling are masked by the secondary effect of drugs like cycloheximide.
We believe that 5PSeq offers a new and complementary window to study global ribosome dynamics in conditions where the use of methods requiring the use of translation inhibition drugs or the isolation of polyribosomal fractions is not possible or advisable.

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High-Throughput Single-Cell Sequencing Library Generation Using Nextera And The LabCyte ECHO 525

Stephan Lorenz
Wellcome Trust Sanger Institute, United Kingdom

Abstract
The advent of single-cell sequencing technologies enabled a new era of biological research. Researchers aim to examine 1,000s of cells to understand the composition and function of cell populations and how individual cells contribute to the greater picture of tissue and organ function, development and disease. Delivering experiments on such scale is a challenging task. Manual execution of the required protocols is laborious and today’s off-the-shelf microfluidic platforms offer limited throughput. By using a combination of flow cytometry, high-speed dispensers, traditional liquid handlers and game-changing acoustic dispensing technology, we established a pipeline that delivers thousands of high-quality single-cell genomes and transcriptomes per day with a turnaround time of 2 days. Economic feasibility was achieved by minimising all assay volumes, in particular library preparation with the Nextera protocol. A conventional Nextera tagmentation and NGS library generation reaction is performed in a 50µl reaction that costs £70. By using acoustic dispensing devices, such as the ECHO 525, a scale-down by 50 – 100x can be achieved with minor modifications of the protocol, leading to significant cost reduction without sacrificing data quality. Furthermore, we demonstrate the superiority of contact-free liquid transfers in molecular biology applications with regard to reproducibility and contamination prevention. Finally, we demonstrate how acoustic dispensing can be integrated with other technologies, such as multicolour flow cytometry, to deliver next-generation experiments.

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Customized Solution Hybridization Enrichment Panels: From A Single Gene To An Entire Exome- Design, Usage, And How To Increase Your Odds For A Successful Outcome

Scott Rose
Integrated DNA Technologies, United States of America

Abstract
While Next Generation Sequencing technology has matured to the point that entire genomes can be sequenced at an improved cost and shorter time period, there is still a very strong demand for the ability to carry out focused sequencing runs with multiple samples completed in a narrow time window. One way to accomplish this is to use custom or pre-developed and validated solution hybridization enrichment panels to selectively target only regions of interest. This way, valuable NGS machine reads are devoted to generating the depth of coverage needed, and increasing the number of samples that can be simultaneously run. This talk will cover the technical issues facing a researcher in using any hybridization based enrichment panel (custom or pre-designed), from initial design, key determinants in setting up a rapid 4 hour hybridization reaction, and how to simultaneously enrich and capture multiple libraries. Based on recent improvements in the field of hybridization enrichment, data will also be shown on what is currently achievable. In addition, practical solutions for improving or altering existing panels without having to reorder a whole new panel will be described.

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Targeted resequencing and variant validation using pxlence PCR assays

Steve Lefever1,2, Frauke Coppieters1,2, Daisy Flamez3, Jo Vandesompele1,2
1Ghent University, Center for Medical Genetics Ghent, Ghent, Belgium; 
2pxlence, Dendermonde, Belgium; 
3Ghent University, Biomarked, Ghent, Belgium

Abstract
Targeted resequencing has become an important application in clinical diagnostics. A wide range of target enrichment approaches are available, enabling the customer to focus on regions of interest. This not only reduces sequencing costs per sample but also facilitates downstream data analysis considerably. Due to its flexibility in design, high sensitivity and specificity, the polymerase chain reaction (PCR) is particularly well suited as enrichment strategy.
We developed and validated a primer design tool to generate one million PCR assays for both fresh frozen and formalin fixed paraffin embedded (FFPE) samples, covering over 99% of the human exome. Assays were designed to generate equimolar and specific amplification using uniform PCR conditions. Several proof-of-concept studies using the pxlence assays have been published. NGS gene panels were developed for congenital blindness (16 genes), deafness (15 genes) and various cancer types (16 genes). Uniform sequencing coverage has been achieved using different library preparations and sequencing instruments (GS FLX, Roche; GAII, MiSeq, Illumina). To date, the Center for Medical Genetics in Ghent uses the pxlence assays in a high-throughput singleplex enrichment workflow to replace Sanger sequencing-based diagnostic tests with NGS.
Due to the excellent performance of both the design pipeline and the generated primer pairs, a spin-off called pxlence (pronounced ‘pixellence’) was recently founded. Its short-term goal is to provide customers easy access to the predesigned assays, enabling them to enrich any exonic region or confirm any variant of interest through either NGS or Sanger sequencing. More information is available at www.pxlence.com

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Next-Generation RNA-Seq

Gary Schroth
Illumina, United States of America

Abstract
A well done RNA-Seq experiment can provide the most comprehensive, accurate and unbiased way to study gene expression, alternative splicing, RNA variation and RNA structure. The past few years have seen amazing technological advances that have led to a wide range of improvements in the RNA-Seq experimental process. Next-Generation RNA-Seq studies are unbiased, accurate, and sensitive, yet work well with low amounts of total RNA (even if the RNA is highly degraded or comes from FFPE samples). These new methods are also less expensive, require less hands-on-time, and are easier to perform than ever before. Finally the data analysis bottleneck associated with RNA-Seq studies has been completely removed with the advent of pipelines that take full advantage of massively parallel cloud computing resources. In this talk I will review the state-of-the-art in RNA-Seq experimental design, library prep, sequencing and data analysis.

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