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Thursday, March 17 


Sponsored by
Epicentre Biotechnologies
7:45 am Breakfast Presentation
Eliminating the Library Preparation Bottleneck

Nick Caruccio, Ph.D., Director, Market Development, EPICENTRE Biotechnologies

EPICENTRE® offers streamlined solutions for DNA and RNA NGS sequencing library preparation. For DNA-Seq, Nextera™ technology is fast becoming the method of choice for library preparation, requiring less input and yielding coverage and complexity comparable to standard methods. In addition, EPICENTRE has recently developed new methods for rRNA removal, and for preparing directional RNA-Seq and small-RNA-Seq libraries compatible with multiple sequencing platforms. Improvements and novel derivatives of DNA-Seq and RNA-Seq library preparation methods will be described.

 

 

Target Capture and Enrichment 

8:30 Chairperson’s Remarks

Olivier Harismendy, Ph.D., Assistant Project Scientist, Moores Cancer Center, University of California, San Diego

8:35 Targeted Sequencing in Cancer Genomics

Olivier Harismendy, Ph.D., Assistant Project Scientist, Moores Cancer Center, University of California, San Diego

The identification of cancer somatic mutations via targeted sequencing of exons offers great opportunities for biomarker research and advanced clinical care. We will present both whole exome sequencing and candidate genes deep sequencing approaches to identify somatic mutations in clinical samples and will discuss their implications for basic as well as translational research.

9:10 Large-Scale Identification and Functional Characterization of lincRNAs in Embryonic Stem Cells

Mitchell Guttman, Ph.D., Graduate Research Assistant, Biology, Broad Institute of MIT and Harvard

We developed a novel statistical reconstruction approach that allows accurate reconstruction of a mammalian transcriptome without prior gene models. Using this approach along with RNA-Seq data in mouse ES cells we identified the gene structures of hundreds of conserved large intergenic non-coding RNAs (lincRNAs). Using these sequences, we designed and validated shRNAs targeting >200 lincRNA genes and systematically characterized their function in the cell. Using this data we can reconstruct the regulatory role of lincRNAs in ES cells.

Sponsored by
Beckman Coulter
9:45 SPRIworks - An Automated Sample Preparation System for Second Generation Sequencing Platforms 

William Donahue Ph.D., Manager, Molecular Biology, Beckman Coulter Genomics
The emergence of second generation sequencing technologies has enabled scientists to generate vastly increased data sets from their sequencing experiments. The dramatic increase in throughput however is supported by workflows that are relatively complicated and labor intensive. The SPRIworks sample preparation systems I, II and III enable automated sample preparation for each platform. Data will be shown highlighting the capability of SPRIworks for sample multiplexing along with its applicability to other workflows such as RNA-Seq and ChIP-Seq.

10:00 Networking Coffee Break in Exhibit Hall with Poster Viewing

Sponsored by
NuGen 
10:45 Sample Preparation Workflow for Large Scale Studies using RNA-Seq
Vincent Magrini, Ph.D., Research Assistant Professor, Department of Genetics & Senior Group Leader, Technology Development, The Genome Center, Washington University School of Medicine
Many of the RNA samples processed by our group are of clinical origin, and present challenges for analysis by next-generation sequencing. Samples collected by biopsy, or preserved by FFPE or LCM techniques are often in low abundance or degraded. After evaluating a number of RNA-Seq technologies for performance by ribosomal RNA content, gene representation, and coverage breadth, we adopted the NuGEN Ovation System for a clinical resequencing projects. For the analysis of human microbiome samples, we are currently evaluating a similar technology from NuGEN for Prokaryotic RNA-Seq.

11:20 Noncanonical Transcripts in Yeast and Human Surveyed Using 3’-End RNA-Seq

Oh Kyu Yoon, Ph.D., Postdoctoral Fellow, Molecular & Cell Biology, University of California, Berkeley

As assay costs decrease and as analysis tools improve, the cost benefit curve will shift and permit the extension of whole exome sequencing to highly informative clinical samples of compromised DNA quality, including paraffin tissue blocks. We will present informative lessons in library construction and acceptable rates of errant calls by split sample strategies, effect of pure versus enriched-cellular tissue samples and cross platform comparisons to examine clinical samples suitable for NGS applications.

11:55 Close of Session

Sponsored by
ROCHE2ROCHE1
12:15 pm Luncheon Presentation
Comparison of Exome Enrichment Platforms and their Application in Mendelian Disease Studies
Rui Chen, Ph.D., Snyder Lab, Department of Genetics, Stanford University School of Medicine

 

Library Preparation 

2:00 Chairperson’s Remarks

Scott Peterson, Ph.D., Professor, Director, Functional Genomics Research Technology, J. Craig Venter Institute

2:05 Extraction and Processing Tissue Samples for NGS

Galen Hostetter, M.D., Associate Investigator, Integrated Cancer Genomics Division; Head, Colorectal Cancer Research; Director, Tissue Microarray Core, TGen

A broad range of genomic and epigenetic assays are increasing performed on NGS platforms as whole genome sequencing and data analysis tools become more cost effective. However, NGS technology cost are significant with considerable time and effort required for library construction and back-end data analysis tools. As assay costs decrease and as analysis tools improve, the cost benefit curve will shift and permit the extension of whole exome sequencing to highly informative clinical samples of compromised DNA quality, including paraffin tissue blocks. We will present informative lessons in library construction and acceptable rates of errant calls by split sample strategies, effect of pure versus enriched-cellular tissue samples and cross platform comparisons to examine clinical samples suitable for NGS applications.

2:40 Rapid Construction of Complex, Low-Input, Low-Bias Fragment Libraries for Massively Parallel DNA Sequencing by Transposase-Catalyzed Adaptor Insertion

Andrew Adey, Graduate Student, Genome Sciences, Molecular and Cellular Biology, University of Washington

Characterization and extension of a low-input alternative library construction method in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. Comparison to conventional methods reveals a slightly greater insertion bias, though this has little impact at the level of genomic coverage. The approach is further applied and expanded on to sequence a human genome to high coverage, as well as detailing alternative protocols for 96-plex sample indexing, sub-nanogram library construction, PCR-free library construction, and direct-from-colony library construction.

3:15 Selected Poster Presentation: Effect of PCR Extension Temperature on High-Throughput Sequencing
Maria Jose Lopez Barragan, Ph.D., Malaria Functional Genomics Section, Laboratory of Malaria & Vector Research, NIAID-NIH
Polymerase chain reaction (PCR) is widely employed to amplify DNA fragments before they are hybridized to a microarray chip or are processed for massively parallel sequencing. DNA regions with high AT content may not be amplified properly or not amplified at all, leading to artificial lack of coverage in AT-rich regions, whereas more GC-rich regions may be excessively amplified. Many organisms such as the human malaria parasite Plasmodium falciparum and free-living protozoan like Paramecium tetraurelia have AT-rich genomes. We investigated the effects of PCR extension temperature on sequence coverage of Illumina parallel sequencing. We constructed three nucleosome libraries using extension temperatures at 60ºC, 65ºC, and 70ºC, respectively, and compared the sequence coverage of the AT-rich regions of the P. falciparum genome. We show that reduction in PCR extension temperature from 70ºC to 60ºC can greatly increase the fraction of coverage at AT-rich regions of the P. falciparum genome. Our method will improve the efficiency and coverage in sequencing an AT-rich genome.

3:30 Networking Refreshment Break in Exhibit Hall with Poster Viewing

4:15 Key Steps for Successful ChIP-Seq Library Preparation

Henriette O’Geen, Ph.D., Project Scientist, Genome & Biomedical Sciences, University of California-Davis

To understand the biology of development, differentiation and disease, it is critical to unravel the underlying transcriptional networks. Chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) is a very effective tool to identify transcription factor binding on a genome wide scale. Using ChIP-seq technology, we investigated the role of GATA2 in vascular biology and discovered a link between GATA2 and inflammation. Protocols used to prepare successful GATA2 ChIP-seq libraries, as well as data analysis, will be discussed.

4:50 Seeing Deeper into Human Microbiomes

Scott Peterson, Ph.D., Professor, Director, Functional Genomics Research Technology, J. Craig Venter Institute

A typical microbiome sample contains a small number of dominant species that are complemented by a very large number of species of moderate and low abundance. The abundance of the species spans at least four orders of magnitude. The application of brute force sequencing therefore provides limited information regarding the majority of species and genomes present. We have implemented a normalization strategy based on Cot analysis that exploits the long-standing observation that abundant nucleic acids will re-anneal faster than DNAs of lower concentration. We will present our findings using this approach for both 16S rDNA profiling and comparative metagenomic DNA analysis focused on twins discordant for dental caries.

5:20 Close of Day



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