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Next Generation Sequencing

 
  May 10, 2011  
     
 
GTC Conference, San Francisco, CA, Hyatt at Fisherman's Wharf
07 -08 Jul 2011


Day 1 - Thursday, July 7, 2011
  
7:00Registration & Continental Breakfast
  
7:55Welcome & Opening Remarks
  
  
 KEYNOTE PRESENTATION
8:00Complete Sequencing Millions of Human Genomes for Research and Genomic Medicine
  
 Rade Drmanac, Ph.D.
CSO and Co-Founder
Complete Genomics
  
 Innumerable biomarkers have been investigated and include DNA, mRNA, microRNA, and proteins. Thus, there are multiple approaches for classifying biomarkers as they relate to the pathogenesis of human disease. Quantitative Fluorescence Image Analysis (QFIA), is a microscope system approach whereby proteins can be precisely quantified in the context of the micro ecosystem of the cellular compartments. Critical to the success of quantification is appropriate sample collection, fixation, instrument calibration, specificity of reagents and etc. Recent advances in optical imaging fluorescent standards and antibody reagents, stable light sources have led to the development of QFIA methodology to study the fundamental events occurring in the cellular actin associated with premalignant and malignant changes associated with human carcinogenesis. The precision of protein measurements has also served to elucidate and confirm the concept of biochemical field disease. Longitudinal studies of workers at risk for bladder cancer have clarified the concept of premalignant field disease and can predict individuals at risk for malignancy years in advance of clinically manifest cancer. The opportunity exists to incorporate this methodology in studying individuals at risk for malignancy and defining specified treatment regimens for the notification of individualized risk assessment, prevention, and therapy. Fundamental concepts of QFIA will be presented for the clinical and research utility of this system.
  
  
  
8:45Richard McCombie, Ph.D., Professor, Cold Spring Harbor Laboratory
  
9:10John Stamatoyannopoulos, Assistant Professor of Genome Sciences, University of Washington
  
9:35Sequencing with Semiconductor Chips
 Maneesh Jain, Vice President, Marketing and Business Development, Ion Torrent
  
 Ion Torrent has invented the first device—a new semiconductor chip—capable of directly translating chemical signals into digital information. The first application of this technology is sequencing DNA. The device leverages decades of semiconductor technology advances, and in just a few years has brought the entire design, fabrication and supply chain infrastructure of that industry—a trillion dollar investment—to bear on the challenge of sequencing. The result is Ion semiconductor sequencing, the first commercial sequencing technology that does not use light, and as a result delivers unprecedented speed, scalability and low cost. 

Ion Torrent sequencing uses only natural (label-free) reagents and takes place in disposable semiconductor microchips that contain sensors that have been fabricated as individual electronic detectors, allowing one sequence read per sensor. The system performance has been demonstrated by sequencing four bacterial genomes, ranging in genomes size and GC content from Vibrio fisheri (4.3Mb genome, 38% GC) to Escherichia coli K12 – (4.6Mb genome, 51%GC) and DH10b (4.7Mb genome, 51%GC) to Rhodopseudomonas palustris (5.5Mb genome, 65%GC). Besides comprising the first genomes sequenced with post-light technology, the genomes are remarkable in the lack of data bias, which is as good, or better, than existing commercial platforms.

Ion’s technological applicability to routine human sequencing has also been demonstrated by utilizing Ion chips to sequence a human genome. We will show how the technology has scaled in just a few months from 1.2 million sensors in the first-generation Ion 314 chips to 6.1 and 11 million sensors in the second-generation Ion 316 and 318 chips respectively—all while maintaining the same 1- to 2-hour runtime. Additionally, Ion has successful accomplished design, manufacture and sequencing using chips possessing the smaller 1-micron diameter well, enabling further increases in well density and sequencing throughput in subsequent chip designs. Because the heart of the system is a novel, disposable sensor, built and assembled using standard semiconductor fabrication methodologies, able to sequence without the need for intermediate enzymes or the constraints of having to image using light, the cost of genome sequencing will continue to fall with each successive generation of denser chips according to Moore’s law.
  
10:00Networking & Refreshment Break
  
10:30Massive Sample Multiplexing: Strategies and Methods
 Corey Nislow, Ph.D., Assistant Professor, University of Toronto
  
 Next Generation sequencing continues to deliver dramatic increases in read number and total sequence/run. These increases decrease the costs of many applications, such as whole genome sequencing, RNA-seq and metagenomics. Interesting, other typically smaller applications require modifications in order to take advantage of these developments. Specifically, experiments that require a few million reads must be multiplexed to be cost effective. The best multiplexing strategies will offer start-to-finish solutions, including barcode design, sequencing protocols and bioinformatic deconvolution. Over the past several years our lab has worked to develop such strategies on all short-read platforms, including Illumina, SOLiD and more recently, Ion Torrent. I will discuss the University of Toronto's Donnelly Sequencing Centre's real-world application of a variety of multiplexing strategies. 

Sample multiplexing:
1. greatly decreases costs and increases throughput
2. can be incorporated into any existing next generation sequencing protocol
3. is platform independent
4. provides genetically linked sample tracking
  
10:55Multiplexing Small RNA Libraries
 Steven Head, Ph.D., Director, Microarray, NGS Core, Scripps Research Institute
  
11:20[Oral Presentations from Exemplary Submitted Abstracts]
  
12:10Lunch On Your Own
  
 FEATURED PRESENTATION
1:30Advances in Next Generation Nucleic Acid Analysis and its Implication in Personalized Medicine
  
 Mostafa Ronaghi, Ph.D.
Senior Vice President and CTO
Illumina
  
 Advances in next-generation sequencing platforms are driving immense growth in biological research. Dramatic cost reduction and comprehensiveness in next generation sequencing have enabled the use of this technology for various biological applications. These include, discovery of new diagnostics and therapeutic markers, as well as cancer care and management of other diseases. In this talk we will review our recent progresses in developing new technologies for integrative genomics. As an example, we will discuss how these technologies are being implemented in cancer care management. 

Highlights in this talk:
1- Isolation of individual cells in biological samples
2- Analysis of single cell with next generation sequencing technologies
3- Recent advances in next generation sequencing
4- Analysis of diverse set of data 
  
2:15Hanlee Ji, M.D., Assistant Professor, Oncology, Stanford University School of Medicine
  
2:40Integration of Omic Data for Prediction of Drug Response in Breast Cancer
 Obi Griffith, PhD, Life Sciences Division, Lawrence Berkeley National Lab
  
 The clinical and genomic heterogeneity of breast cancer necessitates development of personalized therapeutic strategies. Hundreds of new candidate agents are under investigation for use in well-defined patient subpopulations. Cell lines mirror many of the molecular characteristics of the tumors from which they were derived, and are thus a good preclinical model for the study of drug response in cancer. We hypothesize that correlating the responses of a panel of breast cancer cell lines to FDA approved and investigational therapies with their molecular characteristics will reveal biomarkers that can be used to guide clinical trials in terms of patient stratification. A collection of 72 breast cancer cell lines was assembled representing all known molecular subtypes. Cell lines were tested for response to 111 therapeutic compounds by growth inhibition assays. In addition, nine molecular profiling datasets were collected for the cell line panel assessing copy number, expression, transcriptome sequence (RNA-seq), exome sequence, methylation, protein abundance, and mutation status. Classification signatures for drug response were developed using the Random Forests approach. In order to determine the importance of the different molecular data sets, classifiers were built on the data sets separately as well as on the combined data. All data types resulted in optimal response prediction for at least some specific drug compounds. Around one third of drug compounds showed a transcriptional subtype-specific response. For a significant fraction of drugs, the ROC curve (AUC) obtained with molecular data increased by at least 0.1 compared to the predictive power of breast cancer subtype alone.

Benefits

• Describes an approach for integration of multiple omic data types for prediction of response to clinically relevant drugs in breast cancer
• Attempts to assess the relative value of different data types for drug response prediction
• Discusses importance of molecular subtype for prediction of drug response
• Provides example signatures for drug response in breast cancer
  
3:05Networking & Refreshment Break
  
3:40High Throughput, High-Resolution Mapping and Characterization of Autonomously Replicating Sequences in Diverse Yeasts
 Ivan Liachko, PhD, Senior FellowUniversity of Washington
  
 DNA replication initiates at loci termed origins of replication. In yeast, replication origins are short A/T-rich sequence elements (ARSs) that can initiate autonomous replication of plasmids. ARSs have been most well studied in budding yeast where a motif called the ACS is necessary but not sufficient for ARS function. We have developed tools for rapidly isolating and characterizing large numbers of ARSs and have applied our methodology to a number of different yeasts including the industrially important Saccharomyces cerevisiae and Pichia pastoris.

By combining high-throughput versions of classic ARS screening techniques with next-gen sequencing, we have developed ARS-seq – a technique which can yield a comprehensive collection of ARS plasmids within a given species. This collection can be sequenced to identify the locations and sequences of all ARSs in the genome with the average resolution of ~600bp per ARS. In order to localize the essential functional elements within the ARSs, we have developed miniARS-seq, which is used to recover short (~150bp) sub-fragments of ARSs that retain function. In order to identify specific nucleotides important for ARS function, we have developed mutARS-seq, which combines high-throughput mutagenesis and sequencing to screen large numbers of mutant variants of ARSs. In addition, we have developed flankARS-seq, a method of identifying sequences that are able to functionally complement the ACS motif. We have used these techniques to identify and characterize large numbers of ARSs in diverse species of yeast.

• Introduction to yeast replication origins
• Methodology developed to map and characterize origins
• Discovery of novel mechanisms of DNA replication initiation
• Identification of ectopic elements that can function as origins in numerous species of yeast
  
4:05Punctuated Evolution of Breast Tumors Revealed by Single Cell Sequencing
  
 Nicholas Navin, PhD, Assistant Professor, MD Anderson Cancer Center
  
 The prevailing model for human tumor progression assumes that genomes evolve gradually through the accumulation of advantageous mutations in cancer genes over extended periods of time. However, these models are deduced from data that is based on populations of cells, and are thus confounded by complex mixtures in heterogeneous tumors. Here we show that with flow-sorted nuclei, whole genome amplification (WGA), and next generation sequencing we can accurately quantify genomic copy number within an individual nucleus. We apply single nucleus sequencing (SNS) to investigate tumor population structure and evolution in two breast cancer cases. Analysis of 100 single cells from a polygenomic tumor revealed three distinct clonal subpopulations that likely represent sequential clonal expansions. Additional analysis of 100 single cells from a monogenomic primary tumor and its liver metastasis suggested that a single clonal expansion formed the primary tumor and seeded the metastasis. In contrast to gradual models of tumor progression, our data indicate that tumors grow by punctuated clonal expansions with few persistent intermediates. Additionally, we identified an unexpected subpopulation of genomically diverse ‘pseudodiploid’ cells, that may be part of an aberrant process to generate genomic diversity in these tumors.
  
4:30Jingyue Ju, Ph.D., Director, Center for Genome Technology and Biomolecular Engineering, Columbia University
  
4:55Immune Monitoring by High-throughput Sequencing
  
 Scott D. Boyd, M.D., Ph.D., Assistant Professor of Pathology, Director of Sequencing Applications, Stanford University
  
 High-throughput DNA sequencing of rearranged immune receptor loci enables global characterization of human immune responses and lymphoid malignancies. Applications of human immunoglobulin heavy chain (IGH) sequencing include detection of minimal residual lymphoma/leukemia following treatment, tracking the peripheral blood B cells responding to vaccination, and monitoring autoimmune diseases.
  
5:20Networking Reception & Poster Session
  

  
 
Day 2 - Friday, July 8, 2011
  
7:30Continental Breakfast
  
  
 FEATURED PRESENTATION
8:00TBA
  
 Patrice Milos, Ph.D.
Senior Vice President and Chief Scientific Officer
Helicos BioSciences
  
  
  
  
8:35Metallic Nanostructures Provide New Opportunities for Real-Time Free-Running Single Strand DNA Sequencing
 Joseph Lakowicz, Director, Center for Fluorescence Spectroscopy, University of Maryland School of Medicine
  
 We propose the use of plasmonic metal nanostructures for DNA sequencing. As an example we describe their use for free-running single-strand exonuclease sequencing. Exonuclease sequencing has the potential advantages of high-throughput, speed and long read lengths. The use of exonuclease sequencing appears to be limited by the inability to detect and identify single labeled nucleotides in flowing samples. Plasmonic nanostructures can increase the brightness and photostability of fluorophores. We discuss two examples how this phenomenon can be used for sequencing. First, we present free-running high-throughput nanopore sequencing using nanoporous metal films. As an alternative approach, we propose a unique structure of a bi-metallic nano-cylinder with dielectric in between two metal surfaces. Our finite element calculations suggest wavelength selective excitation in a nano-cylinder and four orders of magnitude increase in fluorescence intensity for a silver nano-cylinder. In these approaches, metal optics may be used to increase detection efficiency. Our preliminary experiments and theoretical calculations suggest plasmonic nanostructures provide wavelength resolution. We also describe the possibility of sequencing using intrinsic base fluorescence which is enhanced by nearby metal structures. This approach would eliminate the need for labeling the DNA. The phenomenon of plasmon-controlled fluorescence (PCF) can be used in the present generation of high-throughput sequencers. We believe that the use of PCF will allow design of a new generation of sequencers using simple-to-fabricate nanoporus films with labeled DNA. Additionally PCF may allow sequencing without the need for extrinsic probes.
  
9:00Alexander Balatsky, Scientist, Center for Integrated Nanotechnologies, Los Alamos National Labs
  
9:25Natural DNA Sequencing by Synthesis
 Xiaohua Huang, Ph.D., Associate Professor of Bioengineering, UC San Diego
  
 I will describe our efforts in the development of two DNA sequencing platforms. In one platform called Natural Sequencing by Synthesis (nSBS), a small percentage of a non-terminating fluorescently labeled nucleotide is incorporated along with the natural nucleotide in the cyclic nucleotide-by-nucleotide DNA synthesis process for sequence detection. The sparse incorporation and subsequent removal of the fluorescent labels ensures that DNA synthesis is mostly natural. The decoupling of the sequencing reaction and sequence detection steps allows for greater scalability. Our theoretical modeling and Monte Carlo simulations have shown that homopolymer stretches up to 20 bases long can be sequenced at 99% accuracy with read lengths of 1,000 bases using 10,000 copies of DNA templates. I will present our recent progress in demonstrating the nSBS platform. Natural DNA polymerases can distinguish the 4 different native base types and replicate very long DNA molecules with high fidelity and velocity. In another platform called READS (REAl-time DNA Sequencing) genome technology, we engineer FRET sensors onto the DNA polymerases and use them to monitor the dynamic conformational changes accompanying the incorporation of native unlabeled nucleotides to decode the DNA sequences in real time at the single molecule level. We have been able to monitor the fluorescence from both the donor and acceptor simultaneously during the dynamic chemo-mechanical process of nucleotide binding and incorporation by single DNA polymerase molecules labeled a FRET pair. Our preliminary results indicate that there are observable changes in the FRET signal during DNA synthesis.

Benefits: Learn about: 1) New next-generation sequencing technology; 2) Third- and fourth-generation real time single molecule sequencing technology; 3) Protein engineering; 4) Single molecule microscopy (TIRF, FRET); 5) Microfluidics and instrumentation relevant to sequencing technology development.
  
9:50Networking & Refreshment Break
  
10:30Progress in NGS sequencing application at BGI
 Xu Xun, Ph.D., Chief Executive Officer, BGI Americas
  
 Understanding the huge book of life is the biggest challenge for a biologist. The development of high throughput second/third generation sequencing technologies accelerated the progress and scientists are now showing greater interest and ambition than ever before. Evolutionary history of inter/intra species and development processes of cells differentiates species, individuals and cells. To understand the species tree and understand the biological characteristics of species adaptation, several projects were carried out by BGI in an international collaboration model, which includes the "1000 animal and plant reference genome" project and 10K microbial genome project. As a model in human studies, BGI aims to understand disease, population evaluation and adaptation with projects such as the Tibetan high altitude adaptation project, LuCamp project and the 1000 human genome project. Sequencing the genome of 1000 cancer single cells suggests the driver and passenger genes during cancer progression. High throughput genome sequencing enables us to read the tree of life in multiple scales; from species to individuals as well as at the cell level.
  
10:55Michael Rhodes, Sr. Manager Sequencing Applications, Life Technologies
  
11:20Integrated Data Analysis Pipeline of Bisulfite Sequencing in DNA
 Yuanxin Xi, M.S., Duncan Cancer Center, Baylor College of Medicine
  
 DNA methyaltion is one of the key epigenetic marks in gene expression regulation. Bisulfite sequencing is the most effective technology to detect the methylation status of the genome at single nucleotide resolution. Unmethylated Cytosines were converted to Uracil in bisulfite treatment and sequenced as Thymine after amplification, distinguishing them from the unconverted methy-Cytosine. Accurate and efficient analysis of high throughput bisulfite sequencing data is critical in extracting the biological inferences. However, it remains a great challenge in bioinformatics. Here we present a data analysis pipeline for DNA methyltion studies, which integrates a specifically designed bisulfite reads mapping software, BSMAP and a downstream methylation analysis package mSuite, including functions of methylation ratio estimation, differential methylation region (DMR) detection, pathway analysis and data visualizations. This combined DNA methylation data analysis pipeline is a powerful toolset for processing both whole genome bisulfite sequencing and reduced representation bisulfite dequencing(RRBS). 

Benefits

1. This talk provides a powerful toolset in bisulfite sequencing data analysis in DNA methylation studies
2. This talk also provides a comprehensive overview of the questions and issues in bisulfite sequencing 
3. This talk will also address discuss common challenges in next generation sequencing, i.e. sequencing errors caused by GC content bias in PCR.
  
11:45Lunch
  
1:00Automated Library Preparation Using Digital Microfluidics
 Michael Pollack, Director of Product Development and CTO, Advanced Liquid Logic
  
 Digital microfluidics, characterized by precise and direct manipulation of liquid droplets using electrowetting, is being applied in a variety of applications in research and diagnostics markets. The programmable flexibility of digital microfluidics permits a wide range of protocols to be implemented on a single platform or even on a single cartridge while providing substantial benefits in terms of reagent savings, throughput, ease of use and performance. Advanced Liquid Logic has developed a sample preparation system for Next Generation Sequencing which automates key library preparation steps including end repair, A-tailing, adapter ligation and magnetic bead-based purifications. This system reduces the “hands-on” time for these processes to less than 15 minutes with total process times of less than 3 hours. Compared to conventional methods, the system also reduces reagent consumption by over 90%, significantly reducing the cost per prepared library. The precision and repeatability of digital microfluidics also insures that prepared libraries are of consistently high quality and yield. This flexible open system is designed to work with multiple different reagent vendors and sequencing platforms and new protocols can be readily configured in software. The current status and performance of Advanced Liquid Logic’s library preparation product will be presented along with a discussion of future directions and applications.
  
1:25Denaturation Mapping of DNA in Nanofluidic Channels: A New Approach to Single-Molecule DNA Analysis
 Walter Reisner, Assistant Professor of Pathology, McGill University
  
1:50Stephen Y. Chou, Professor, Princeton University
  
2:15The Application of NGS to Characterizing HIV, HBV, and HCV Drug Resistance
 Robert Shafer, Associate Professor, Stanford University
  
 

HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) are the most prevalent deadly chronic viral diseases. Although HIV is a retrovirus, HBV is a double-stranded DNA virus, and HCV is a single-stranded RNA virus, each has a high mutation rate and exists within individuals as a swarm of innumerable yet related sequence variants. The resulting antiviral drug resistance that often arises compromises the success of available therapies and complicates the development of new antiviral compounds. During the past five years, my laboratory has been using high-throughput “deep sequencing” NGS technologies to characterize HIV, HBV, and HCV variants within treated and untreated infected patients.

The performance and, in particular, the analysis of HIV, HBV, and HCV deep sequencing studies is fraught with challenges that are unique to each virus. My talk will focus on the methods that our laboratory has developed to address these challenges. I will then summarize the clinical significance of our most recent deep sequencing studies designed to detect and thwart the emergence of HIV, HBV, and HCV antiviral drug resistance.

  
2:40Joe Mellor, Research Associate, University of Toronto
  
3:05TBA
  
3:30Conference Concludes
 
 
Organized by: GTC Conference
Invited Speakers:
KEYNOTE SPEAKERFEATURED SPEAKER
  
Rade Drmanac, Ph.D.
CSO and Co-Founder
Complete Genomics
Mostafa Ronaghi, Ph.D.
Senior Vice President and CTO
Illumina
 
 
FEATURED SPEAKER
 
Patrice Milos, Ph.D.
Senior Vice President and Chief Scientific Officer
Helicos BioSciences
 

DISTINGUISHED SPEAKERS
 
Alexander Balatsky
Scientist, Center for Integrated Nanotechnologies
Los Alamos National Laboratory 
 
Scott Boyd, M.D., Ph.D.
Assistant Professor
Stanford University

 
Stephen Chou
Professor
Princeton University

 
Obi Griffith
Lawrence Berkeley National Lab
 
Steven Head
Director, Microarray, NGS Core
Scripps Research Institute

 
Xiaohua Huang, Ph.D.
Associate Professor of Bioengineering
UCSD

 
Maneesh Jain
Vice President, Marketing and Business Development
Ion Torrent

 
Hanlee Ji
Assistant Professor, Dept. of Medicine, Division of Oncology
Stanford University School of Medicine

 
Jingyue Ju, Ph.D.
Director, Center for Genome Technology and Biomolecular Engineering
Columbia University

 
Joseph Lakowicz
Director, Center for Fluorescence Spectroscopy 
University of Maryland School of Medicine 
 
Ivan Liachko
Postdoctoral Associate, Department of Genome Sciences
University of Washington
 
Joe Mellor
Research Associate
University of Toronto
 
Ivan Liachko
Postdoctoral Associate, Department of Genome Sciences
University of Washington
 
Richard McCombie
Professor
Cold Spring Harbor Laboratories
 
Nicholas Navin
Assistant Professor
MD Anderson Cancer Center

 
Corey Nislow
Principal Investigator
University of Toronto
 
Michael Pollack
Director of Product Development and CTO
Advanced Liquid Logic
 
Michael Rhodes
Sr. Manager Sequencing Applications
Life Technologies
 
Robert Shafer
Associate Professor
Stanford University

 
John Stamatoyannopoulos
Assistant Professor of Genome Sciences
University of Washington 

 
Yuanxin Xi
Duncan Cancer Center
Baylor College of Medicine
 
Xu Xun
Chief Executive Officer 
BGI Americas
 
Deadline for Abstracts: 07 Jun 2011
 
Registration: https://www.gtcbio.com/index.php?option=com_register&cn=Next%20Generation%20Sequencing&cid=33
E-mail: infogtcbio@gtcbio.com
 
   
 
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