TUESDAY, MARCH 17

7:30 am Breakfast Presentation  Sponsored by

Advances in MicroRNA Profiling Using Quantitative Real-Time PCR
Iain Russell, Ph.D.,  Senior Product Manager, Applied Biosystems
Applied Biosystems Megaplex™ Primer Pools and TaqMan^® MicroRNA Arrays provide an ideal workflow for the profiling of microRNAs from human, mouse and rat samples, even when only minute amounts of sample (down to 1ng) are available.  Utilizing the same novel stem-loop RT primers and assay design as the individual TaqMan^® MicroRNA Assays, the Megaplex™ profiling workflow is highly sensitive, offers a broad dynamic range (up to 7 logs) and is highly specific, enabling quantitation of only the mature microRNA sequences in as little as 5 hours.  Here we present data demonstrating the high performance of the Megaplex™ workflow and provide specific examples of how it has been applied to real-life biological applications.

8:15 Java and Jive Discussion Groups
Grab a cup of coffee and join a facilitated discussion group focused around specific themes. This unique session allows conference participants to exchange ideas, experiences, and develop future collaborations around a focused topic.  Topics include:

Table One:  Real-Time PCR Data-Analysis
Host:  Jo Vandesompele, Ph.D., Center for Medical Genetics, Ghent University Hospital
Data-analysis is the last part of the gene expression workflow (preceded by sample preparation, assay design and validation, and actual PCR), and is often considered to be the last remaining hurdle. In this session we will discuss effective normalization strategies, quantification models, inter-run variation and calibration, experiment design, error on PCR efficiency estimation, etc.

Table Two:  Taking Microfluidics Mainstream
Host: Reg Beer, Ph.D., Lawrence Livermore National Laboratory
Discussion areas will focus on:

• Microfluidics is making highly multiplexed assays routine, how many is enough?
• Strategies for defeating amplification bias
• What is your ideal sample volume?
• PCR instruments are getting faster, but how fast is fast enough?

Table Three:  Moving from a Research Method to Valid Biomarker
Host:  Nathan O’Callahan, Ph.D., CSIRO, Nutrigenomics/Nutrigentics Laboratory
Discussion topics will focus on steps required and challenges for development of methods to use in monitoring health and well being.

Table Four:   Internal and External Controls for Real-Time PCR Assays
Host:  Ian Kavanagh , Ph.D., R&D Manager, Thermo Fisher Scientific
Discussion areas will focus on:

• What controls are currently being used and what applications are they being used ofr
• What are the advantages and disadvantages of the different approaches to controls
• What improvements can be made to overcome the negative issues

Table Five:    High Resolution DNA Melting
Host:  Carl Wittwer, Ph.D., Professor, Pathology, University of Utah
Discussion points include:

• Mutation scanning
• Amplicon genotyping and unlabeled probe genotyping
• Snapback primers
• Instruments and Dyes 
  

Table Six:  The Future of qPCR - Where are we Going?
Host: Elena Hidalgo Ashrafi, Senior Research Associate , TriLink BioTechnologies, Inc.
We will discuss the limitations of qPCR, including: 

• Increased PCR sensitivity - single cell PCR, ePCR, limiting clinical samples
• Increased PCR efficiency - how to obtain consistent, early Cts
• Improved multiplexing - improved reproducibilty, larger number of targets
• Faster overall reaction time - how well does this translate into reliable results for applications such as Multiplex, one-step RT-PCR, Fast cycling?

Table Seven:  Public Large-scale Long-term Projects Shared Across Multiple Sites
Host: Henrik Bengtsson, Ph.D., Dept of Statistics, UC Berkeley 
Discussion topics:

• With a never-ending continuous stream of samples being processed, when and how often should we reanalyze earlier samples?
• Do we need to utilize databases for storing the data, or is the file system good enough?  The latter is easier to share sites
• How can we assert that the results are reproducible?
• In order to absorb the vast amount of information in high-through pipelines, use case-oriented reports (vignettes) will be generated automatically.  However, the number of such reports will still be enormous.  How do we organize and navigate among these reports?

Table Eight:  Copy Number Analysis
Host: Mike Lelivelt, Ph.D., Vice President, Partek Inc.
Topics to be discussed include:

• Finding Shared Segments
• Overlaying Segments with Annotations
• Integration with other technologies

REAL RESULTS

9:00 Chairperson’s Remarks
Karl Hasenstein, Ph.D., J.J. Chance Professor, Biology, University of Louisiana

9:05 Standardized Quantitative RT-PCR Assays for Quantitation of Yellow Fever and Chimeric Yellow Fever-Dengue Vaccines
Nathalie Mantel, M.S., Virology Unit Leader, Research, Sanofi Pasteur
Yellow fever-dengue chimeras (CYDs), are currently being developed as live tetravalent dengue vaccine candidates. Specific quantitative assays are needed to evaluate the viral load of each serotype in vaccine batches and biological samples. We established a quantitative real-time RT-PCR (qRT-PCR) system comprising five one-step qRT-PCRs targeting the E/NS1 junction of each chimera, or the NS5 gene in the yellow fever backbone. Each assay was standardized using in vitro transcribed RNA qualified according to its size and purity, and precisely quantified. A non RNA-extracted virus sample was introduced as external quality control (EQC), as well as two extraction controls consisting of two doses, 40 and 4000 GEQ (Genomic Equivalents), of this EQC extracted in parallel to the samples. Between six and ten GEQ/reaction were reproducibly measured with all assays and similar titers were obtained with the two methods when chimeric virus samples were quantified with the E/NS1- or the NS5-specific assays. Reproducibility of RNA extraction was ensured by automation of the process (yield ≥50%), and infectious virus was isolated in ≥ 80 % of PCR-positive sera from immune monkeys.

9:35 Challenges of qPCR Analysis Using Samples From Human Post-Mortem Brain
Elizabeth Thomas, Ph.D., Assistant Professor, Molecular Biology, The Scripps Research Institute
Quantitative real-time PCR analysis remains the best way to accurately measure gene expression changes under different conditions or in response to various stimuli. Such techniques are commonly used to investigate molecular pathologies related to neuropsychiatric disorders, especially in the context of validating microarray expression data. However, studies using human, post-mortem CNS tissue present several challenges which will be discussed, including RNA quality issues, demographic data of human subjects and choice of reference genes for normalizing qPCR data.

10:05 High-Throughput qPCR Assay to Validate siRNA-Mediated Gene Knockdown in Large Scale Samples
Yaping Liu, M.S., MCIT, Research Scientist, Automated Biotechnology, Merck & Co., Inc
For the purpose of validating siRNA-mediated gene knockdown in large scale samples generated from genome-wide siRNA high throughput screening (HTS), we have developed a high-throughput Taqman qPCR assay using cell lysates made from transfected cells in 384-well format. The process overcomes the bottleneck of RNA purification and increases throughput significantly. An automated workflow and sample data from siRNA HTS projects will be introduced.

10:35 Coffee Break, Poster & Exhibit Viewing

11:00 A Quantitative Real-Time PCR Method for Absolute Telomere Length
Nathan O’Callaghan, Ph.D., CSIRO – Human Nutrition, Genome Health, Nutrigenomics and Nutrigenetics Laboratory
Telomere shortening is an important risk factor for cancer and accelerated aging. We have developed a simple, robust and reproducible method to measure absolute telomere length. This method, based on Cawthon’s qRTm- PCR assay, uses an oligomer standard that is able to generate absolute telomere length values, rather than relative quantification. We have demonstrated a strong correlation between this improved method and the ‘gold standard’ of telomere length measurement, TRF by Southern hybridisation. The capability to generate absolute telomere length values should also allow a more direct comparison of results between experiments, within and between laboratories.

11:30 Targeted Exome Re-sequencing: Comparative Analysis of Two Sample Enrichment Processes by Sequence Capture Microarray and Long-Range PCR Followed by Next Generation Sequencing (NGS)
Lan-Szu (Bob) Chou, Ph.D., Scientist, Research and Development/Molecular Sequencing and Genetics, ARUP Laboratories
To attempt to implement NGS in diagnostic laboratories for targeted disease gene(s) re-sequencing and address the issues of (1) sample enrichment processes, (2) data management, and (3) reagent package cost, we investigated a DNA sample enrichment process using a customized high density oligonucleotide microarray to enrich targeted regions of the NF1 gene (17q11.2). We then evaluated possible software solutions for data management using 454 data (GS FLX). Finally, we estimated the relative cost per run and the feasibility to incorporate NGS workflow into the diagnostic laboratory setting. In results, functional NF1 sequences were successfully captured and enriched, with the unique matching reads to the NF1 locus in an average of 58%. For reference-mapping assembly purposes, the Windows based software seemed to be powerful enough, which helps to facilitate the implementation of NGS into laboratories relying on Windows applications. To decrease the cost per targeted region sequenced, running pooled samples labeled with unique sequence tags is a potential option.

12:00pm Close of Session

12:15 Luncheon Presentation Sponsored by

Expression Profiling of CTCs using Digital PCR on the Fluidigm Platform
Daniel Shoemaker, Ph.D., Chief Scientific Officer, ICx Biosystems
We are using Digital PCR on the Fluidigm platform to measure subtle changes in gene expression in CTCs. The method is highly quantitative and generates reproducible expression data down to the single cell level. A key advance has been using DNA targets to normalize the expression data to an exact “transcripts per cell” output.

SEQUENCING TECH EXPO

Next-Generation Sequencing is alive, thriving, and driving discovery. As costs come down and ease increases, these new, massively parallel high-throughput sequencing platforms are infiltrating multiple aspects of traditional biological research. However, each next-generation sequencing platform best lends itself to specific sequencing goals. This Tech Expo showcases the next-generation sequencing platforms to help you make informed purchasing decisions.

Sponsored Seminars Hosted by:

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

2:05 The SOLiD™ 3 System - Taking Next-Generation Sequencing to the Next Level  
Michael Rhodes, Ph.D., Product Applications Senior Manager, Genetic Analysis, High Throughput Discovery, Applied Biosystems
The new SOLiD™ 3 System achieves new milestones in throughput in excess of 20 Gb of mate paired sequence data from a single run and 30-40 Gb of demonstrated throughput in Applied Biosystems R&D labs. Maintaining high accuracy, improvements in read length and unique mate-pair library strategies, the news system enables expanding applications from whole genome resequencing and SNP discovery to miRNA profiling. This presentation will review various applications that the new system capabilities enable and discuss an example of whole genome transcript profiling in single cells and whole genome resequencing for SNP and structural rearrangement discovery.

2:35 Enabling True Biology with Helicos™ Single Molecule Sequencing
Patrice M. Milos, Ph.D., VP & CSO, Helicos BioSciences Corporation
Helicos True Single Molecule Sequencing (tSMS)™ provides a unique view of genome biology through the direct sequencing of cellular nucleic acids in an unbiased manner providing both quantitation and sequence information. Using a simple DNA sample preparation which requires no ligation or PCR amplification genomic DNA is sheared, tailed with polyA and readied for hybridization to a flow cell surface containing oligo dT for initiating the sequencing by synthesis reactions. To demonstrate the fidelity and scale of the HelicosTM Genetic Analysis System three bacterial genomes were sequenced, each of distinctly different genomic contents in single flow cell channels of the available 50 channels. We have extended our research to include a variety of genomic targets including candidate gene regions, yeast and C. elegans, all with similar accuracy and coverage. The ability of our single molecule sequencing platform to provide quantitative measurements of genome biology include research efforts in small RNA measurements, assessment of copy number variation of human samples and a simple method for quantitative assessment of the transcriptome, digital gene expression – all without the requirement of ligation or amplification – a hallmark for measuring the biology of cells.

3:05 Refreshment Break, Poster and Exhibit Viewing

3:30 The Illumina Genome Analyzer - Transforming Systems Biology
Abizar Lakdawalla, Ph.D., Senior Product Manager, Sequencing Applications, Illumina
The Genome Analyzer next-generation sequencing system has transformed our understanding of genome variations, epigenomics, transcriptomics, and the interaction of proteins with DNA and RNA. A comprehensive description of the Genome Analyzer system will be presented with effective approaches to address broad systems biology questions. Strategies and tools derived from sequencing multiple human genomes, large numbers of transcriptomes, and extensive ChIP-Seq samples will be described to maximize the data and sample throughput with the simple and easy-to-use Genome Analyzer workflow.


4:15 Moving Next Generation Sequencing into the Clinical Research Market:
Timothy Harkins, Ph.D., Director, 454 Sequencing Roche Applied Science, Roche, Inc
The Genome Sequencer FLX is now generating the longest reads within the next gen market with over 1 million unique sequencing reads that are 400 to 500 base-pairs in length. With a fast instrument run time of 10 hours and the ability to quickly analyze the sequencing data, projects involving 1,000’s of samples are able to be processed readily. The projects that will be presented include:
1) Sequencing HIV to detect low frequency drug resistant mutations
2) HLA sequencing – the most known polymorphic regions within the human genome
3) Using NimbleGen Sequence-Capture arrays to sequence the whole human exome
4) Detecting novel pathogens in complex environmental samples


4:45 Interactive Panel Discussion with Sequencing Leaders
Moderator; Kevin Davies, Ph.D., Editor-in-Chief, BioIT World
The term “next-generation” has become the“now generation.” As the genome unit price of these next-gen platforms continues to tumble, excitement is growing about the scientific and commercial potential of third-generation sequencing systems, from single-molecule methods to nanopores to ‘nanoball’ service models. Here, leaders from established and emerging next-gen platform providers trade insights on the latest scientific and technological advances, and answer your questions..

5:30 Close of Meeting