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Exploring Next Generation Sequencing - Day 3



7:30 am Breakfast Technology Workshop Sponsored by SGI Intel
Sequencing Data to Results: Application of Optimized Computational Tools
Deepak Thakkar, Ph.D., Director of Marketing - Higher Education, BioSciences, National Labs, sgi
Next generation genome sequencing technologies are revolutionizing the genomics workflows and presenting complex computing and data management challenges. Not only must scientists contend with an overwhelming volume of data, today’s economic competitive environment requires that they complete their work in less time.  As the amount of data collected grows, fully understanding and leveraging the data to advance research may well become the bottleneck in R&D productivity.  Along with large data volumes, heterogeneous data formats have added to the complexity of analyzing data to make scientific decisions. This is especially true in biosciences, which is at the nexus of bioinformatics, chemistry and classic biology. Optimal computing platforms for analyzing these complex workflows and then providing access to the data in a complex enterprise are a key challenge. Advances made in these areas will be discussed in form of case studies.

8:15 Successful Sequencing Discussion Groups
Time has been designated for facilitated discussion groups with specific themes. This unique opportunity allows conference participants to focus on a topic and exchange ideas, information, experiences, and develop future collaborations.

Setting Up a Next-Generation Sequencing Lab Individual User Experiences

9:00 Chairperson’s Remarks

9:05 Pennsylvania State University
Mammoth Genomics
Stephan C. Schuster, Associate Professor, Center for Comparative Genomics and Bioinformatics
Next-generation sequencing in its initial phase was limited by shorter read-length than the more mature Sanger capillary-technique. While this is considered a shortcoming by some, its bias-free sequencing without any cloning or amplification biases made it the method of choice for environmental or ancient DNA projects. This is in particular true as ancient DNA is restricted in a post- mortem degenerative process to short fragments that matches the read-length of the currently available technology. We will describe the use of the sequencing-by-synthesis approach for the analysis of the mammoth genome. In recent studies we were able to demonstrate that it is now possible to generate large amounts of sequence data from the nuclear genome of an extinct animal. Using the draft sequence of the extant African elephant we distinguish between reads that are endogenous from environmental reads. As our approach also produces mitochondrial reads in large numbers, we can assemble complete mitochondrial genomes with high redundancy. This allows for the identification of DNA damage alleles and therefore for the distinction between DNA damage and sequencing errors. Our recently published technique to isolate DNA from ancient mammoth hair has allowed for the fast and economic analysis of multiple individuals. It is therefore now possible to study the population structure of an extinct species using the resolution of complete mt genomes.

9:35 Harvard Partners
Illumina’s Next-Generation Sequencing Platform as the Flexible Tool for Screening siRNA Libraries
Oleg Iartchouk, Ph.D., Director, Sequencing Technologies and Mutation Detections, Center of Genetics and Genomics and Broad Institute 
The Center of Genetics lab has developed several approaches for customized sequencing of pooled shRNA libraries which should expand the choices for applications for next-generation sequencing scientists.  The presentation will highlight the customized primers and protocols that were used to optimize the quality of the sequencing.

10:05 Technology Spotlight  Sponsored by RaindanceTechnologies
Maximizing the Efficiency of Targeted Re-Sequencing Using Droplet-Based Microfluidics

Jeffrey Olson, Project Lead, Nucleic Acid Applications, Raindance Technologies
In order to exploit the full potential of new sequencing techniques, a robust method for isolating biologically relevant genomic loci on the megabase scale is required. This technique has been commonly referred to as “genomic selection” or “targeted re-sequencing.” RainDance Technologies has developed a unique solution to address the key bottleneck in targeted re-sequencing process. By leveraging advancements in droplet-based microfluidics, the platform provides the capability to amplify a theoretically unlimited number of loci using high-throughput PCR in droplets while avoiding limitations traditionally associated with multiplex amplification or hybridization methods. We present the application of the droplet-based microfluidics technology to selectively amplify target loci while minimizing selection bias and maximizing the efficiency of the overall targeted re-sequencing workflow. We will also show examples of the benefits of improved sequencing efficiency for analysis of sequence variation for studies involving large numbers of samples.

10:20  Morning Coffee

10:50 MIT/Whitehead Institute for Biomedical Research
Using SNPs for Measuring microRNA-mediated mRNA Destabilization
Jinkuk Kim, Graduate Student, David Bartel’s Lab, Health Science and Technology
We first identified heterozygous SNPs in animal tissues that create or destroy microRNA target sites. The gene that possesses such SNP will have one allele with a microRNA target site and the other allele without one. Then, we measured imbalance in allelic expression by RT-PCR amplifying the mRNA fragments containing the SNPs, multiplex-sequencing (454 pyrosequencing) the PCR products, and counting the number of reads from each allele. When examining 67 SNPs, we observed that the allele with a target site tends to be downregulated compared to the other allele without one and that the degree of downregulation is dependent on the type and context features of target sites. Our approach can be applied to testing the functionality of endogenous microRNAs and studying other classes of cis-regulatory motifs.

11:20 Yale University
Mapping Regulatory Regions Across Genomes: Chromatin and Transcription as Viewed by Next-Generation Sequencing

Ghia Euskirchen, Ph.D., Associate Research Scientist, Yale University Center of Excellence in Genome Sciences  
Vast territories of the human genome have yet to be explored for functional domains. Regions both proximal and distal to genes are expected to be important for normal cellular function and genetic stability. Massively parallel sequencing has allowed unprecedented opportunities to map functional regions of the human genome and to understand basic principles of gene regulation. Examples will be presented from a number of these studies as well as direct experience gained from generating, analyzing and managing next-generation sequencing data.

11:50 Close of Session

12:00 Luncheon Technology Workshop (Sponsorship Available) Or Lunch on Your Own

Driving Discoveries

2:00 Chairperson’s Remarks
Kevin Davies, Ph.D., Editor-in-Chief, BioIT World

An Integrated Data Warehouse for Biomedical and Translational Research
John Quackenbush
John Quackenbush, Ph.D., Professor, Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute 
The critical components of modern biomedical research are patient samples and their associated clinical data. However, in most research environments, the data is scattered across many fragmented and often incompatible databases. Similarly, there exists a vast body of public data, including sequence and gene expression repositories, the biomedical literature, and countless other sources of information that, though available, are often not readily accessible for large-scale automated analysis. To address these issues and accelerate research, we have developed a data warehouse and a series of data portals that, driven by common research use cases, provide an integrated picture of biomedical research data.

2:40 Absolute Transcript Counting by Single Molecule Sequencing      Sponsored by Helicos 
Tal Raz, Ph.D., Group Leader/Senior Scientist, Methods Development, Helicos Biosciences
We have developed a transcript counting method using single molecule sequencing to generate unbiased and accurate quantification for the complete transcriptional dynamic range of typical cells. Our method is not subject to the typical biases inherent to most digital gene expression (DGE) profiling. In addition, we apply a computational method for eliminating counting inaccuracies that may be caused by read misassignment. We demonstrate the application of single molecule transcript counting that requires no ratiometric measure between samples for accurate inventorying of the complete transcriptome of a saccharomyces cerevisiae mRNA and a human placenta mRNA. We compare our results to microarray and qPCR measurements and demonstrate the ability to quantify low-abundance transcripts.

3:00 Sponsored by Febit
Targeted  Extraction of Specific Non-Contiguous Loci on Mouse Chromosome 1 for Next-Generation Sequencing with HybSelectTM 

Daniel Summerer, Ph.D., Head of Application Development, Enzyme-on-Chip Technologies, febit, gmbh
The introduction of next-generation sequencers has lead to a dramatic increase in sequencing throughput. Besides whole genome de novo sequencing of small genomes, high-throughput resequencing of selected regions of a large, eukaryotic genome is now available. However, these selected regions have to be separated from the remaining chromosomal DNA first due to the high complexity of the sample. Methods like PCR amplification are time-consuming and very expensive. We will report an approach termed HybSelect™ which makes use of target-specific DNA extraction using Geniom® Biochips. Up to 120.000 capture probes specific for the genomic region of interest are synthesized on the Biochip and hybridized with the sample which can be analyzed via next-generation sequencing after washing and elution.  The advantages of using this extraction method will be also be presented in addition to the targeted enrichment of a set of 100 non-contiguous loci on the mouse chromosome 1.

Next-Next Generation Sequencing Technologies

3:20 Shotgun Sequencing by Hybridization: Simple, High-Throughput and Low Cost
Benoit Houle, Ph.D., Senior Director, Technology Development, Genizon BioSciences

3:50 Conference Wrap-Up

4:00 Close of Meeting



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