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RNA-Seq Experimental Design and BioinformaticsGenetic Privacy: Technology and EthicsMicrobes and Human Health: The What, Where, How and Why
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As prices continue to drop and technology continues to improve, the purchase of a next-generation sequencing (NGS) platform is now a reality for most research laboratories. Once you have purchased a platform, however, how do you maximize the greatest potential for your investment? Realizing this potential requires efficient workflow strategies, careful experimental design, comprehensive targeted enrichment technologies, data analysis, management and integration, in addition to maintaining your platform and people management all at maximum production. The central theme at CHI's Sequencing Strategies for Success conference is efficient utilization of your NGS platform. Sessions will focus on common bottlenecks, case studies, real-world experiences and solutions from experienced NGS users.
Day 1 | Day 2
Tuesday, August 20
7:30 am Breakfast Technology Workshop (Sponsorship Opportunity Available)
Tools, Tips 'n Tricks
8:15 Chairperson's Remarks
Mats Ljungman, Ph.D., Associate Professor, Radiation Oncology, University of Michigan
8:20 Functional Analysis of the Nascent Transcriptome Using "Run-On" Sequencing Methods: Looking Beyond the Pipeline
Hojoong Kwak, M.D., Research Scientist, Molecular Biology and Genetics, John Lis Laboratory, Cornell University Biography
We have recently developed a series of nascent RNA sequencing that identifies the active RNA polymerase with extreme sensitivity and up to base pair resolutions named GRO-seq and PRO-seq. First-pass processing of the "run-on" sequencing data uses the common "pipelines" established for RNA-seq or ChIP-seq. We present a few of our next-level analyses of various functional aspects of RNA polymerase machinery, such as the high-resolution pausing patterns and elongation rates.
8:50 RNA-Seq for Enrichment and Analysis of IRF5 Transcript Expression in SLE
Betsy J. Barnes, Ph.D., Associate Professor, Biochemistry and Molecular Biology, New Jersey Medical School University Hospital Cancer Center, University of Medicine and Dentistry New Jersey Biography
This presentation will discuss enriched RNA-seq for the rapid identification and quantification of differentially spliced transcript variants in primary immune cells from genotyped healthy donors and patients with systemic lupus erythematosus (SLE). This technique enabled the identification of an IRF5 transcript signature that associates with patients carrying the IRF5-SLE risk haplotype.
9:20 Bru-Seq and BruChase-Seq
Mats Ljungman, Ph.D., Associate Professor, Radiation Oncology, University of Michigan Biography
Bru-Seq and BruChase-Seq are based on the metabolic pulse-chase labeling of nascent RNA with bromouridine followed by isolation of Bru-labeled RNA and deep sequencing. While Bru-Seq informs us on the relative rates of RNA synthesis genome-wide, BruChase-Seq assesses the kinetics of splicing and the relative stability of all transcripts. These techniques have revealed an astonishing wealth of information about the complexities of expression of coding and non-coding RNA in human cells as well as splicing kinetics, elongation rates, transcription start sites and transcription from enhancer elements.
9:50 Selected Oral Poster Presentation: Cross-Contamination Monitoring Using a Unique Sequence Spike-In Control
Richard A. Moore, Ph.D., Sequencing Group Leader, Genome Sciences Centre, British Columbia Cancer Agency
As sample numbers have increased, and clinical re-sequencing has developed, it has become essential to include quality control measures to exclude compromised samples. This requires accurate identification of cross-contamination and sample swaps throughout the pipeline, as well as close monitoring of sample identity. We have developed a panel of unique DNA sequences cloned into a common vector (>1000 verified). These are added to the sample when it is first received and can be detected with a simple PCR and sequencing from any stage of the process. Sequence reads derived from this spike-in are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. Successful spiking directly into blood and tissue samples prior to DNA extraction has been demonstrated. This methodology has been implemented in our targeted clinical diagnostic pipeline and has also been successfully validated for whole-genome sequencing. It is a very inexpensive, flexible and platform-agnostic solution to a pressing need.
10:05 Coffee Break in the Exhibit Hall with Poster Viewing
11:00 Using Multiplex ChIP-Seq to Explore Centromere Evolution in Budding Yeast
Philippe Lefrancois, Ph.D., Faculty of Medicine, University of Montreal Biography
We applied multiplex ChIP-Seq to characterize Saccharomyces cerevisiae kinetochore proteins directly and indirectly binding to chromatin, in multiple genotypes. In this organism carrying 126-bp, point centromeres, we discovered regions with centromeric activity elsewhere in the genome when the levels of a centromeric histone are elevated. These novel regions, sharing characteristics of both point and larger regional centromeres, have important implications for the evolution of centromeres.
11:30 Design and Analysis of CLIP-Seq Experiments for Identifying Targets and Functions of RNA-Binding Proteins
Anastasios Vourekas, Ph.D., Research Scientist, Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania Biography
In vivo crosslinking of RNA-protein complexes and high-throughput sequencing of fragments of these RNAs is revolutionizing the way RNA-binding proteins are studied. This method provides transcriptome-wide landscapes of RNA targets bound in vivo with nucleotide-level resolution. I will present guidelines and examples for the design and data analysis of CLIP-seq experiments and how these can lead to a comprehensive understanding of the functions of diverse RNA-binding proteins.
12:00 pm Rapid Assembly and Analysis of Clinical Sequence Data: Using DNASTAR Software to Identify Disease-Causing Mutations
Matthew Keyser, Next-Gen Applications Scientist, DNASTAR, Inc.
DNASTAR offers an integrated suite of software for assembling and analyzing clinical sequencing data on a desktop computer. In this example, we align exome data from a disease and control group to the human genome using DNASTAR software. After alignment, the same software package is used to view the results and identify SNPs and genes of interest.
12:30 Close of Session
12:30 From Reads to Variants: Ten-Fold Reduction in Time and Cost with Improved Accuracy
Rupert Yip, Ph.D., Director, Product Marketing, Bina Technologies
Alignment and variant calling of raw NGS reads has been plagued by expensive HPC hardware and the bioinformatics personnel to support and maintain home-grown, open-source secondary analysis solutions. Such solutions can take up to weeks and $1000s per analysis. We present a genomic analysis platform that reduces, by ten-fold, the time and cost for secondary analysis while improving accuracy compared to standard pipelines. Our innovative model reduces costs by ten-fold while preventing hardware obsolescence.
» Plenary Keynote Session
2:00 Chairperson's Opening Remarks
Toby Bloom, Ph.D., Deputy Scientific Director, Informatics, New York Genome Center
2:10 A Revolution in DNA Sequencing Technologies: Challenges and Opportunities
Jeffery A. Schloss, Ph.D., Director, Division of Genome Sciences, National Human Genome Research Institute, National Institutes of Health Biography
The initial sequencing of the human genome spurred an appetite for much more human sequence information to better understand the contributions of human sequence variation to health and disease. However, despite dramatic reductions during the Human Genome Project, the cost of sequencing was clearly too high to collect the very large numbers of human and numerous other organism genome sequences needed to achieve that understanding. In 2004, NHGRI launched parallel programs to reduce the cost of sequencing a mammalian genome initially by two (in five years), and eventually by four orders of magnitude (in ten years). This presentation will summarize the technologies that are in high-throughput use to produce stunning amounts of sequence and related data and novel biological insights, and will emphasize technologies currently emerging and on the horizon that may provide human genome sequence data with the nature, quality, cost and turnaround time needed for applications in research and medicine.
2:50 RNA is Everywhere: Characterizing the Spectra and Flux of RNA in Mammalian Circulation
David Galas, Ph.D., Principal Scientist, Pacific Northwest Diabetes Research Institute Biography
The discovery of foreign RNA in blood and tissues of humans and mice raises many questions, including its origins, the mechanisms of its transport and stability and what, if any, functions it has. I will discuss what we know about circulating exRNA in human plasma and the use of NGS in the exploration of this new area of investigation in biology and medicine.
3:30 Refreshment Break in the Exhibit Hall with Poster Viewing
4:15 Genomics and the Single Cell
Sherman Weissman, Ph.D., Sterling Professor of Genetics and Medicine, Yale University School of Medicine Biography
Studies of single cells are being approached by widely different methods, principally either florescence microscopy including super-high resolution methods, cloning and expansion of single cells or most generally applicable, genomic-scale nucleic acid analyses. The last includes single-cell DNA sequence analysis, gene expression analysis and most recently analyses of telomere length, DNA methylation and potentially closed regions of chromatin. Also, in the near future, it may be possible to combine several analyses of a single cell, including mRNA expression, genomic DNA methylation and protein secretion. These approaches will have major value for diverse fields, including molecular analysis of the early stages of development, the nature and heterogeneity of stem cells and transient repopulating cells in various systems including the hematopoietic system, the nature and extent of heterogeneity of neurons, heterogeneity in neoplasia and in functional subsets of cells of the immune system. A substantial experimental challenge is to distinguish technical variation from stochastic and deterministic events in single cells. Another, broader challenge is to correlate the results of genomic properties that necessarily involve destruction of the cell with the functional properties and potential of the individual cell being analyzed. These issues will be discussed briefly in the presentation.
4:55 Genome Hacking
Yaniv Erlich, Ph.D., Principal Investigator, Whitehead Fellow, Whitehead Institute for Biomedical Research Biography
Sharing sequencing datasets without identifiers has become a common practice in genomics. We developed a technique that uses entirely free, publicly accessible Internet resources to fully identify individuals in these studies. I will present quantitative analysis about the probability of identifying U.S. individuals by this technique. In addition, I will demonstrate the power of our approach by tracing back the identities of multiple whole-genome datasets in public sequencing repositories.
Genetic Privacy: Technology and Ethics with Yaniv Erlich
5:35 Close of Sequencing Strategies for Success Conference / Short Course Registration Open
6:00-9:00 Dinner Short Course*
SC3: Assembly and Alignment
* Separate Registration Required
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