7:30 am Breakfast Technology Workshop (Sponsorship Available)

8:30 Chairperson’s Remarks

8:35 High-Resolution Analysis of Structural Variation in the Human Genome
Lars Feuk, Ph.D., Senior Research Associate, Center for Applied Genomics, Hospital for Sick Children, Toronto
We are working towards a high-resolution map of copy-number variation of the human genome. The methods employed include a combination of high-resolution comparative genomic hybridization (CGH), alignment of human sequence assemblies and analysis of sequence read-depth. We will report on the discovery of new variants and comparison of the different approaches for variation discovery.

9:05 Charting and Sequencing Structural Variation Using High-Resolution Paired-End Mapping (HR-PEM)
Jan Korbel, Postdoctoral Fellow, Molecular Biophysics and Biochemistry Department, Yale University
Structural variation (SV), i.e. deletions, duplications, insertions, and inversions involving kilo- to Megabases of genomic DNA, has recently been suggested to be responsible for a considerable amount of phenotypic variation, and possibly, disease in humans (1-6). However, to date, most methods for identifying structural variants are low resolution (7) (>50 kb) and fail to precisely identify the boundary sequences (i.e. breakpoints) of SVs. We present a novel approach, High-Resolution Paired-End Mapping (HR-PEM), which makes use of 454/Roche sequencing technology, and combines computational analysis, high-throughput PCR assays, and amplicon-cocktail-sequencing to rapidly identify SVs at high resolution, and subsequently sequence across their breakpoints. The approach involves sequencing the ends of circularized 3 kb genomic fragments and mapping them onto the human genome reference sequence. We have used HR-PEM to map and sequence SVs ­ i.e. simple deletions, insertions, and inversions, as well as more complex events ­ in two individuals in order to generate a precise map of SVs and their associated breakpoints. From 21 million and 10 million end-pair sequences from each individual, several hundred SVs have been predicted so far, ranging from 2kb to several Mb in size. A first pass PCR analysis indicates that at least 60% of the predicted SVs can be amplified in a single PCR band and analyzed using DNA sequencing. Our results reveal an unexpected level of structural variation in the human genome, and suggest mechanisms by which this layer of variation in the genome has arisen.

9:35 Identification of Complex DNA Variants in a Single Human
Samuel Levy, Ph.D., Senior Scientist, Human Genomic Medicine, J. Craig Venter Institute
Employing a novel, high-throughput, random shotgun sequencing, assembly, variant detection and haplotype assembly approach we have generated diploid genome sequence of a single human donor. Using newly developed genome assembly strategies and comparative genome-to-genome mapping methods it was possible to identify a large number of non-SNP variants that comprise >74% of all variant DNA bases in the donor genome. These findings permit a detailed and personal depiction of the donor's genetic contribution and a preliminary glimpse into our understanding of personal biology.

10:05 Coffee Break, Poster and Exhibit Viewing

10:30 Real-time DNA Sequencing
Susan H. Hardin, Ph.D., President and CEO, VisiGen Biotechnologies, Inc.
VisiGen Biotechnologies, Inc. is developing a sequencing platform that will enable comprehensive genome analysis. VisiGen scientists have engineered polymerase and nucleotides to act together as direct molecular sensors of DNA sequence information during DNA synthesis. As a nucleotide is incorporated into the nascent DNA strand, energy transfers from the excited donor fluorophore attached to the polymerase to the acceptor fluorophore bonded to the nucleotide¹s gamma-phosphate, thereby stimulating the emission and detection of a base-type-specific signature. Because the acceptor fluorophore is naturally removed during nucleotide incorporation, VisiGen’s strategy enables real-time DNA sequencing. The technology is scalable: these nanosequencing machines are monitored in massively parallel arrays to produce a sequencing platform that will be capable of collecting sequence data at rates approaching 1 million bases per second.

10:55 Low Cost Instrument for Next Generation Sequencing
by Synthesis
Steven Gordon, Ph.D., CEO, Intelligent Bio-Systems, Inc.
Intelligent Bio-Systems’ next generation sequencing platform is based on proprietary sequencing by synthesis chemistry invented at Columbia University. The system utilizes single-stranded DNA fragments as templates and incorporates specially labeled nucleotide analogs into a growing complementary second strand one base at a time. Four different dye colors are used to indicate the identity of the nucleotide (A, C, T or G) that is incorporated in each cycle. A compact, cost-effective bench-top instrument which will be capable of producing several gigabases of sequence in per day will be available over the next few months. In this talk we describe several unique features of the technology that enable rapid and low cost data generation. The sequencing by synthesis chemistry uses nucleotide analogs with proprietary reversible terminators and cleavable dyes to produce high-precision, low noise data cycle after cycle. The high-density chip uses a compact array of millions of spots of amplified DNA fragments and the instrument combines fluidics, fluorescent microscopy and software control to completely automate the chemistry and detection cycles. Since the instrument will be at least half the cost of other sequencing instruments, it is expected that many labs outside large genome centers will be able to perform entire genome studies.

11:20 Polony Multiplex Analysis of Gene Expression (PMAGE) and Mouse Hypertrophic Cardiomyopathy
Jonathan Seidman, Ph.D., Professor, Department of Genetics, Harvard Medical School
PMAGE (for "polony multiplex analysis of gene expression"), detects messenger RNAs (mRNAs) as rare as one transcript per three cells. PMAGE incorporates an improved ligation-based method to sequence 14-nucleotide tags derived from individual mRNA molecules. Using PMAGE, we identified early transcriptional changes that preceded pathological manifestations of hypertrophic cardiomyopathy in mice carrying a disease-causing mutation. PMAGE provided a comprehensive profile of cardiac mRNAs, including low-abundance mRNAs encoding signaling molecules and transcription factors that are likely to participate in disease pathogenesis.

1:30 Chairperson’s Remarks

1:35 Deep Sequence Analysis of Clinically Relevant env Region Sequence Variants from HIV
Marilyn Lewis, Principal Scientist E-Biology, Biomarkers and Translational Biology, Pfizer Ltd.

2:05 Deep Sequence of HIV RT Regions in Search of Drug Resistance Mutations in Patients Undergoing RT Inhibitor Treatment
E. Randall Lanier, Ph.D., Senior Investigator, Department of Virology, GlaxoSmithKline
The relationship between virologic failure of anti-HIV regimens and the emergence of resistance is not well understood. Do low abundance resistant variants at baseline reliably predict virologic failure? Does failure with "sensitive" virus mean resistance is not a cause or consequence of virologic failure? Deep sequence analysis allows novel exploration of these questions.

4:00 Software Tools for Polymorphism Discovery in Next-Generation Sequencer Data
Gabor Marth, Assistant Professor, Biology, Boston College
Next-generation, super-high throughput short-read sequencers produce orders of magnitude more bases than capillary sequencers at a fraction of the cost, replacing traditional sequencing platforms for resequencing applications. The main informatics challenges for using short-read sequencer data for polymorphism discovery are (1) to produce accurate base sequence and base quality information; (2) to identify which regions of a genome are unique enough to be resequenced with a given sequencing technology and read length; (3) to align tens or hundreds of millions of reads efficiently and accurately; (4) and to update existing polymorphism discovery programs or write novel programs that can analyze the vast amount of new sequencer data. We have developed a suite of software tools to address these challenges: a base caller program, PYROBAYES, for 454 sequencer data; a general sequencer reads re-aligner / assembler program, MOSAIK; a short-read polymorphism detection program, POLYBAYES++, and a new alignment viewer, EAGLEVIEW. We report the application of our pipeline for SNP and INDEL discovery, mutational profiling, copy number variation detection, and the analysis of transcriptome resequencing data.

4:30 The High-Coverage Genome Sequence of a Single Human
David A. Wheeler, Ph.D., Associate Professor, Human Genome Sequencing Center, Baylor College of Medicine
Recent advances in DNA sequencing using a combination of genomic DNA shearing, limited dilution, and single molecule-primed amplification by emulsion PCR and a massively-parallel method of sequencing in picoliter size reaction vessels, provide the reduced cost and increased speed to enable the generation of data for ‘personalized genome sequencing’. With this technology, we produced a 6X coverage sequence of the genome of James D. Watson. This talk will present the analysis and characterization of genomic variation from this single human DNA sequence.

5:00 ENCODE Pilot Project and the Future of Comparative Sequence Analysis
Elliott H. Margulies, Ph.D., Investigator, Genome Technology Branch, Head, Genome Informatics Section, National Human Genome Research Institute, National Institutes of Health
Identifying sequences under purifying selection can provide clues about the positions of functional elements in the human genome. However, our understanding of the relationship between evolutionary sequence constraint and genome function has been hampered by a lack of synchronized sequence and experimental datasets for the same genomic regions. The ENCyclopedia Of Dna Elements (ENCODE) Consortium is providing this coordination, bringing together investigators with diverse experimental and computational approaches to comprehensively analyze the same 1% of the human genome. Using a high-confidence set of evolutionarily constrained annotations, we established correlations with experimentally identified protein-coding genes, non-coding transcribed regions, sites of protein-DNA interactions, promoter activity, and regions of open chromatin. While most of the experimental annotations are significantly enriched for constrained sequences, we note that large portions of each class of experimentally identified element (with the exception of protein-coding sequences) show no evidence of constraint during mammalian evolution. These conclusions suggest that other more sophisticated approaches for analyzing evolutionary sequence constraint are needed to fully understand how functional sequences in the human genome have evolved.

5:30 Close of Conference