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THURSDAY, MARCH 19
Sponsored by7:30 am Breakfast Presentation
Implementation of Fluidigm’s SlingShot Absolute Quantitation Product Offers the Dual Advantage of Replacing the Costly and Time-Consuming Titration Step as Well as Recovery of Sub-Optimal DNA Samples for 454 Next Generation Sequencing
Joseph Boland, Ph.D., M.S., Dedicated Scientific Operations Leader, SAIC-Frederick, Inc., The National Cancer Institute
We have successfully implemented Fluidigm’s SlingShot technology into our 454 sequencing process at two key junctions: sub-optimal sample quantitation and replacement of the library titration. This implementation has allowed us to process previously failed samples (Roche recommends samples be between 3ug and 5ug) and replace the costly titration step saving us processing time (up to 2 days) plus the cost of a sequencing reaction.
Our initial experiment utilizing SlingShot consisted of 12 samples ranging in amounts from 990ng to 2.5ug. We processed these 12 samples using the approved Roche 454 protocol through library prep. After completion of the library prep, each sample was quantitated using the SlingShot product to accurately assess the amount of sample to go forward into bulk emulsion prep. Due to limitations of the existing quantitation methods available at the core genotyping facility (Ribogreen and Agilent), these samples would have never made it to the sequencer. We will present data highlighting the quality coverage attained (each sample had at least 20X coverage) from these samples using the quantitation obtained through SlingShot. We will also discuss future experimentation as well as future updates we would like to see with this platform.8:15 Successful Sequencing Discussion GroupsGrab a cup of coffee and join a facilitated discussion group focused around specific themes. This unique session allows conference participants to exchange ideas, experiences, and develop future collaborations around a focused topic. Topics include:
Table 1: Keeping Up with a Next-Gen Sequencer: Alternatives to Owning a Giant Computer ClusterHost: Martin Gollery, CEO, Tahoe InformaticsDiscussion focus topics include:
Table 2: Getting Most of Your Illumina Genome Analyzer IIHost: Iwanka Kozarewa, Ph.D., Sequencing Technology Development, Wellcome Trust Sanger InstituteThe discussion will be focused on key steps in sample preparation procedure using Illumina and sequencing flow as well as on some of the most promising applications of next-generation sequencing. Discussion topics include:
Table 3: Next Generation Storage NetworkingHost: Jacob Farmer, CTO, Cambridge ComputerTopics include:
Table 4: Deep Sequencing of Small RNAs in hESC: Exploring Epigenetic Networks in DevelopmentHost: Ron Hart, Rutgers University, and Loyal Goff, CSAIL & Broad, MITDiscussion to include:
Table 5: Pyrosequencing to Pipeline DevelopmentHost: Alla Lapidus, Ph.D., Group Leader, Joint Genome Research InstituteTopics include:
Table 6: Complete Genomics: Rapid, Accurate, Affordable Human Genome Sequencing Host: Radoje Drmanac, Ph.D., Co-founder and Chief Scientific Officer, Complete GenomicsDiscussion topics include:
Table 7: Clinical Utilities of the Next-Generation Sequencing Technology: Issues Before Implementation in Routine LaboratoriesHost: Bob Chou, Ph.D., Scientist, Research and Development/Molecular Sequencing and Genetics, ARUP Laboratories
Discussion topics include:
Table 8: Using magnetic beads in Sample Prep Applications for SequencingHost: Patrick J. Finn Ph.D., Director of Research and Development, Agencourt Bioscience
Clean sample preparation is important to any successful application. Using high performance paramagnetic bead-based technologies immobilizes nucleic acids onto magnetic microparticles. This technology has set an industry standard in sample preparation for Sanger sequencing where products, such as the the Agencourt® CleanSEQ® and Agencourt AMPure® product lines, have been essential reagents for labs engaged in high throughput Sanger sequencing. With the emergence of second generation sequencing systems such as Roche’s 454 FLX, Illumina’s GAII and Life Technologies SOLiD system, the this technology is re-emerging as a key reagent for library sample prep upstream of each sequencing system.
All trademarks are property of their respective ownersDATA MANAGEMENT AND ANALYSIS
9:00 Chairperson’s RemarksMichael Rhodes, Ph.D., Product Applications Senior Manager, Applied Biosystems
9:05 Gene Wiki: Community Intelligence Applied to Gene Annotation
Andrew Su, Ph.D., Group Leader, Computational Discovery, Genomics Institute of the Novartis Research Foundation
There are ~25,000 genes in the human genome, and evidence shows we’ve only scratched the surface in terms of describing their function. It is becoming increasingly clear that the research community cannot solely rely on curators at annotation authorities to annotate genes in databases. Therefore, following broader trends in information management, we present two efforts to harness the power of community intelligence. First, the”Gene Wiki” enables the scientific community to collaboratively summarize gene function in real time. Second, BioGPS offers a gene annotation portal that the bioinformatics community and data providers can collaboratively develop and extend.
9:35 Assembling Short and Error-Prone DNA Reads: Does the Read Length Matter?
Mark Chaisson, Ph.D., Postdoctoral Scholar, Computer Science Engineering, University of California, San Diego
Current sequencing technologies attempt to maximize read length without sacrificing base calling accuracy. Since fragment assembly with inaccurate reads is difficult, the common practice is to trim the inaccurate tails of the reads. We present EULER-USR tool for assembling short reads that bypasses the problem of limited accuracy in the tails of reads. An important and counterintuitive implication of this result is that one may extend sequencing reactions “past their prime”’ to where the error rate grows above what is normally acceptable for fragment assembly. We compare EULER-USR with other short read assemblers and illustrate its applications for assembling mate-paired Illumina reads. We further use EULER-USR to study the question “Does the read length matter?” and to compute the “read length barrier”. We show that increasing the read length beyond the read length barrier does not improve the quality of assembly.
10:05 Estimation of Evolutionary Parameters, and Design of Association Studies Using Resequencing Data
Paul Marjoram, Ph.D., Assistant Professor, Preventative Medicine, Keck School of Medicine, University of Southern California
We will demonstrate how data from next-generation sequencing technologies can be used to estimate evolutionary parameters (mutation and recombination rates), despite the fact that the data does not necessarily provide complete sequence information. We will also present two refinements of our methods: one that is more robust to sequencing errors and another that can be used when no reference genome is available. Finally, we will discuss the implications of these technologies on the design of association studies, and how the power of such studies varies with differing levels of coverage.
10:35 Refreshment Break and Poster Viewing11:00 Poster Awards Sponsored by
11:10 Next-Generation Sequencing of Humans
Pauline Ng, Ph.D., Senior Scientist, Genomic Medicine, J. Craig Venter Institute
We are now in an exciting era where individual human genomes and large genomic regions from human populations can be sequenced. This has been made possible by next-generation sequencing technologies such as Roche 454, ABI SOLiD, and Illumina GA. We have applied NGS technologies to an individual human genome and to population-based sequencing. We discuss the performance of these technologies and how this has improved our understanding of what it will take to achieve a $1,000 genome.11:30 End-User Presentation: Next-Gen Sequence Data Analysis Reveals Grapevine VirusMaher Al Rwahnih, Ph.D., Department of Plant Pathology/FPS, University of CaliforniaThe informatics infrastructure needed for aligning and annotating next-generation reads against reference sequence data presents a daunting challenge for researchers. Plant pathologists at the University of California, Davis are facing a novel grapevine disease that they suspected may be caused by a previously uncharacterized RNA virus. They sampled and sequenced the overall RNA content of the cells of both diseased and apparently healthy grapevines to generate about 400,000 reads with an average length of about 200 nucleotides. Pairwise comparisons would create tens of trillions of alignments, taking weeks on a typical computer, creating an intractable situation.
This case study will demonstrate how these UC Davis researchers overcame computational and analysis limitations to annotate, assemble, and contig the next-gen reads and ultimately identify a new virus, using On-Demand Informatics that minimized cost and delivered timely results.
12:00 Close of Morning Session
12:15 Luncheon Presentation Sponsored by Targeted Resequencing by Agilent in-Solution Genome Partitioning in Combination with the Illumina Genome Analyzer Sequencing PlatformLira Mamanova, Ph.D., Sequencing Technology Development Group, Wellcome Trust Sanger InstituteNext-generation DNA sequencing technologies have greatly increased the throughput of DNA sequencing and have drastically reduced the cost. In spite of this, however, it is still not feasible to perform whole genome sequencing of large numbers of samples, and so it is desirable to be able to isolate specific regions of interest prior to sequencing. Several approaches to sequence capture are available to enrich the specific genomic fragments for targeted resequencing. Here we present an evaluation of the Agilent SureSelect™ Target Enrichment System, where specific regions of a genome are selected by hybridization to biotinylated RNA-oligonucleotides in solution, in combination with the Illumina Genome Analyzer sequencing platform.Utilizing Agilent’s SureSelect Technology for Resequencing Projects on the Illumina GA PlatformRyan Tewhey, Division of Biological Sciences, University of California, San Diego Scripps Genomic Medicine, The Scripps Research InstituteWhile next generation sequencing platforms have greatly increased our ability to analyze millions of bases, the need still exists to enrich for sequences of interest. We have applied Agilent’s SureSelect enrichment technology, a solution based hybridization method, to both DNA isolated from cell lines and clinical blood samples. We have evaluated the technology’s ability to both design probes and subsequently uniformly capture over 3.8 Mb of targeted sequence. The ultimate goal of individual sequencing is the discovery of variants, therefore we have also evaluated the ability of the SureSelect technology to efficiently capture both alleles in comparison to known genotypes.
Sponsored by12:45 Luncheon PresentationThe Next-Next-Generation of
Bioinformatics -Maximizing the Value of Unprecedented Data Growth
Ram Appalaraju, Ph.D., Vice President of Product Marketing and Development, Isilon Systems
The data growth and performance needs of DNA sequencing are changing rapidly and unexpectedly, driven by a recent flurry of next-generation technologies. With research possibilities veritably limitless, a critical challenge remains – where to store this deluge of data and how best to maximize its potential for creating future biomedical breakthroughs. The solution? Scale-out, file-based storage architectures have emerged as an ideal solution for powering bioinformatics research, delivering the performance, scalability and ease of use necessary to allow researchers to focus on their science and not their storage. This session will focus on: Advances in DNA sequencing and other bioinformatics-related technologies driving unprecedented data growth; The implications of this data growth for researchers and scientists, as well as its potential to transform modern medicine; Scale-out, file-based storage architectures as the most compelling solution for maximizing the potential of bioinformatics data.
2:00 Chairperson’s Remarks
Kevin Davies, Ph.D., Editor-in-Chief, BioIT World
2:05 Case Study One: Metagenomics as a New Challenge for Next-Generation Sequencing Technologies
Ludmilla Chistoserdova, Research Scientist, Chemical Engineering, University of Washington
Alla Lapidus, Ph.D., Group Leader, Joint Genome Research Institute
Sequencing DNA of entire microbial communities, known as metagenomics, along with the downstream meta-approaches, metatranscriptomics and metaproteomics, present incredible opportunities for analyzing the genomes of uncultivated microbes. The two major challenges in metagenomics, compared to analyzing individual genomes, are the significantly increased complexity of the DNA complement and unequal abundance of DNA of different organisms. Thus the success of metagenomics will rely on the significantly increased sequence coverage that next-generation sequencing technologies offer, but will ultimately depend on the availability of tools allowing reliable assembly, organismal binning and annotation. We use a highly complex microbial community inhabiting Lake Washington sediment, as a model, to test the feasibility of the currently available sequencing and analysis tools.
2:50 Case Study Two: Whole Transcriptome Sequencing of Cancer Biopsies for Concurrent Analysis of Expression,
Splicing and Mutation
Trevor Pugh, B.Sc., Genome Sciences Centre, BC Cancer Agency
Ryan Morin, B.Sc., M.Sc., Genome Sciences Centre, BC Cancer Agency
Clinicians routinely obtain tumor biopsies for diagnostic purposes, making them an appealing source of DNA and RNA. The amount of RNA that can be obtained from some biopsies is often too small to enable the construction of libraries for next-generation sequencing. We have tested multiple RNA amplification strategies to enable robust production of RNA-seq libraries from biopsies. The paired reads are aligned to the human reference genome supplemented with a set of exon-exon junction sequences. The number of unambiguous reads deriving from individual genes, exons, and exon-exon junctions are counted, providing a digital measure of gene expression and splicing. This provides an opportunity to compare the expression of genes and their inclusion of individual exons between tumor and normal samples. In addition to gene expression changes, we are also able to identify SNPs and somatic mutations in expressed genes. The ability to confidently identify mutations is affected by individual gene expression, sampling depth, and the amount of “end bias” in a given library. Using this approach, we have identified novel somatic mutations in a lobular breast cancer, as well as candidate mutations in multiple biopsies from lymphomas and lung adenocarcinomas.
3:35 Case Study Three: Novel MicroRNA Gene Prediction in Human Enbryonic Stem Cells and Neural Precursors
Ronald Hart, Ph.D., Professor, Keck Center for Collaborative Neuroscience, Rutgers University
Loyal A. Goff, Ph.D., Postdoctoral Associate, Computer Science and Artificial Intelligence Laboratory, The Broad Institute @ MIT
4:20 Case Study Four: Tools to Analyze Metagenomics: A Cystic Fibrosis Case Study
Rob Edwards, Ph.D., Department of Biology, San Diego State University
Dana Hall, Ph.D., Department of Biology, San Diego State University
5:05 Close of Meeting
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