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FRIDAY, MARCH 19, 2010
7:00 am Registration Open
7:30 Breakfast Presentation (Sponsorship Opportunity Available)
8:30 Chairperson’s Remarks
Gary P. Schroth, Ph.D., Sr. Director, Illumina
8:35 Whole Genome Analysis of mRNA Degradation
Timothy Read, Ph.D., Associate Professor, Medicine/ Infectious Diseases & Human Genetics, Emory University School of Medicine
RNA-Seq experiments primarily measure accumulation: information on RNA degredation is necessary to calculate changes in mRNA transcription rate. For two strains of the model bacterium Bacillus cereus with drug-abrogated transcription, we used Illumina GAII and 454 data to derive the kinetics of RNA degradation across the genome. This allowed us to detect sequence variation that influenced stability of orthologous genes, as well as determine the direction of mRNA degradation, map operon structures, and detect patterns of multicistronic mRNA degradation.
9:05 Transcriptome Sequencing of the Microarray Quality Control (MAQC) RNA Reference Samples Using Next-Generation Sequencing
Roderick Jensen, Ph.D., Professor, Biological Sciences, Virginia Polytechnic Institute
9:35 A Digital Gene Expression Study of Endocrine Disruption in Fish from Southern California Coastal Waters
Gary Hardiman, Ph.D., Associate Professor, Department of Medicine; Director, Biomedical Genomics Microarray Facility, University of California, San Diego
A multi-gene cross species microarray was fabricated as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. This provided a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species. Gene-specific expression in liver tissue was measured by qRT-PCR and RNA seq and correlated to microarray data. These analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared to those from non-impacted areas. These studies are providing valuable de novo transcriptome sequence information that can be correlated with known pollutants and fish physiologic data and are uncovering novel biomarkers for development of improved diagnostic tools such as the multi-gene cross-species microarray.
10:05 Networking Coffee Break & Final Poster Viewing
Poster Competition Sponsored by 
10:45 Transcriptome-Wide Profiling for Normal Breast Tissues
Min Li, Ph.D., Field Application Specialist, Partek, Inc.
Yunlong Liu, Ph.D., Assistant Professor, Biostatistics, Center for Computational Biology and Bioinformatics, Indiana University
The breast is one of the most complex genetic organs within the body. This is because the expression levels of its genes are under the control and influence of the hormonal milieu present in the circulating plasma, which changes as a function of age; and for premenopausal women as a function of the menstrual cycle. The goal of this project is to characterize molecular variability of breast tissues from volunteer donors with no clinical evidence of breast malignancy. Normal breast tissues were procured from the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. In order to eliminate bias from stromal tissue, normal samples were laser capture microdissected for ductal cells and RNA extracted from the excised tissue. Libraries were prepared for the Applied Biosystems (ABI) SOLiD3 sequencer using the Whole Transcriptome Analysis Kit from ABI. Herein we present a preliminary analysis of the transcriptomes of normal breast tissues from two individuals. A multitude of analysis is ongoing, including but not limited to differentially expressed genes, identification of novel transcripts, and alternative splicing.
11:15 RNA-Seq/CNV-Seq: A New Method to Detect Copy Number Variation (CNV) using High-Throughput Sequencing
Xie Chao, Ph.D. Candidate, Department of Biological Sciences, National University of Singapore
Copy Number Variation is an important class of genomic variation. Presented will be a method we have developed to detect CNV using next-generation sequencing and CNV-seq. This method is based on a robust statistical model that describes the complete analysis procedure and allows the computation of essential confidence values for detection of CNV.
11:45 Panel Discussion with Morning Speakers
12:15 pm Close of Session
12:30 Luncheon Presentation Sponsored by 
Scaling up Exome Sequencing with a Lab and Data Management System
Johanna Swanson, Data Analyst, Scientific Programmer, University of Washington
Mike Sanders, Product Manager, GenoLogics
A recent laboratory information management implementation at the University of Washington, enabled Dr. Mark Rieder's Northwest Genome Center to quickly scale up high-throughput next gen exome sequencing. A focus on sample integrity while the lab innovated procedures and methods drove a challenging but successful project. Information for samples, containers, reagents, robotics, QC, and results are tracked from sample submission to sequencing. The approach, design, key hurdles and lessons learned will be discussed, as well as how sample tracking aids in running accurate experiments.
(Combined Session with Quantitative PCR)
2:00 Chairperson’s Remarks
Lawrence J. Wangh, Ph.D., Professor, Biology, Laboratory of Molecular Medicine & Global Health, Brandeis University
2:05 Microfluidic Technologies for Single Cell Genetic and Expression Analysis
Yong Zeng, Ph.D., Postdoctoral Researcher, Chemistry, University of California, Berkeley
Single cell analysis is the new frontier for advancing our understanding of molecular mechanisms underlying cellular function and dysfunction. We will first discuss a fully integrated bioprocessor that enables sensitive single-cell gene expression profiling by integrating the capture of single cells, reverse transcriptase (RT)-PCR, product purification, and electrophoretic analysis. Second, a high-throughput single cell genetic analysis (SCGA) technique will be presented that combines a novel microfluidic droplet generator for encapsulation of single cells in nanoliter emulsion droplets with quantitative digital genetic detection via multiplex PCR. These techniques allow us to resolve a distinct bimodal expression of the GAPDH gene in single Jurkat cells treated by siRNA knockdown, and to achieve extremely low-level detection of one E. coli O157 pathogen cell in a background of 105 E. coli K12 cells.
2:35 Strategies using LATE-PCR to Detect and Analyze Sequence Variations in Target Molecules
Lawrence J. Wangh, Ph.D., Professor, Biology, Laboratory of Molecular Medicine & Global Health, Brandeis University
LATE-PCR is an advanced form of asymmetric PCR that efficiently generates abundant single-stranded amplicons. LATE-PCR reactions can readily be multiplexed and quantitatively analyzed at end-point using low temperature probes. The accumulated amplicons can also analyzed by Dilute-’N’-Go sequencing. Application of these technologies to analysis of sequence variations will be discussed using examples of bacterial genes and mitochondrial genes.
3:05 Single Molecule Closed Complex DNA Sequencing
Ernest W. Kovacs, Ph.D., Bioorganic Chemist, GE Global Research
GE Global Research is working to develop a new method of DNA interrogation that can be used for high throughput, single-molecule DNA sequencing. The proposed method utilizes novel nucleotide analogues that have a detectable tag attached to the terminal phosphate. We have found that in the absence of divalent cation, DNA polymerase can be frozen in a stable ternary complex (a “closed complex”) consisting of the DNA primer-template, the DNA polymerase, and the incoming nucleotide as encoded by the DNA template. This complex is “frozen” in the midst of a nucleotide incorporation event, and can be interrogated using single molecule fluorescent microscopy to determine the identity of the trapped base. After visualization, the complex is chased with divalent cation to allow incorporation of the trapped base, and the process repeated over and over to generate the DNA sequence.
3:25 A Strategy for High-Quality Clinical Resequencing of the Human Genome
Hanlee Ji, M.D., Assistant Professor, Oncology, Stanford University
3:45 Prospects for Nanopore-based Direct DNA Sequencing
Andrew D. Hibbs, Ph.D., CEO, Electronic Bio Sciences LLC
Passing a single unamplified strand of DNA through a small pore and reading the signal of individual bases by a direct electrical measurement, without intermediate reactants or consumables, approaches the limit of simplicity for sequencing. Controlling the random motion of the DNA as it passes through the pore is emerging as a critical factor to enable the ultimate success of the method. The basic issues and technology elements involved in such a measurement will be presented, and the remaining key scientific issues discussed.
4:05 Panel Discussion
4:30 Close of Conference
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