WEDNESDAY, MARCH 18
7:00 am Registration and Morning Coffee
Sponsored by7:30 Breakfast Presentation
Targeted Sequencing Using Droplet-Based Microfluidics
James Brayer, Manager of Commercial Scientific Applications, RainDance Technologies, Inc.
Recent advances in DNA sequencing technologies have improved accuracy and dramatically reduced the cost of DNA sequencing. However, even with the improved efficiency of these second-generation systems, sequencing thousands of whole human genomes across various phenotypes is expensive and time consuming. In order to exploit the full potential of these new targeted sequencing techniques, a robust method for isolating biologically relevant genomic loci on the megabase scale will be required. The Sequence Enrichment application from RainDance Technologies leverages the sensitivity and specificity of PCR in a novel droplet-based format which avoids the limitations of traditional multiplex amplification or hybridization methods. The session will focus on: Key aspects of the targeted sequencing workflow that directly impact the quality of the data; An introduction to the RainDance RDT 1000 instrument and Sequence Enrichment application; A discussion of how the Sequence Enrichment process integrates into the targeted sequencing workflow for both short-read and long-read sequencing platforms; and example data from targeted sequencing of thousands of genomic loci with a focus on completeness, uniformity and specificity.
8:30 Chairperson’s Remarks
8:35 KEYNOTE PRESENTATION
Richard Gibbs, Ph.D., Director and Professor, Human Genome Sequencing Center, Baylor College of Medicine
TARGETED SAMPLE ENRICHMENT
9:15 SCODA Electrophoresis for Biomolecule Concentration
Andre Marziali, Ph.D., Director, Department of Physics and Astronomy, University of British Columbia
We have developed a novel electrophoretic concentration technology for efficiently purifying and concentrating biomolecules. The technology offers unique advantages in biomolecule concentration including exceptional rejection of PCR inhibitors, an unparalleled ability to enrich for low abundance nucleic acids, and ability to length-select nucleic fragments. Additionally, we will present recent advances in the SCODA (Synchronous Coefficient of Drag Alteration) technology, including reduction of sample processing times to less than ten minutes and applications to protein concentration.
9:45 Probing the Epigenome of Induced Pluripotent Stem Cells with Methylome Partitioning
Kun Zhang, Ph.D., Assistant Professor, Department of Bioengineering, University of California San Diego
DNA methylation is one of the primary epigenetic regulatory mechanisms that involve in normal developmental processes. We have recently developed a method for specific capture of an arbitrary subset of genomic targets using padlock probes, and for digital quantitation of DNA methylation at the single nucleotide resolution. We used this method to characterize the changes of DNA methylation during the de-differentiation of human fibroblasts into induced pluripotent stem (iPS) cells and pluripotent hybrid stem cells. We found that the phenotypic changes during the reprogramming are associated with local and specific alteration of methylation close to a small set of genes.
10:15 Targeted Extraction of Specific Non-Contiguous Loci on Mouse
Chromosome 1 forSponsored by Next-Generation Sequencing with HybSelectTM
Michelle Lyles, Ph.D., Vice President of Marketing and Sales, 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 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. Geniom® Biochips offer several advantages over regular DNA microarrays, including highly flexible probe content and a unique microfluidic architecture, enabling complete automation of the process and minimizing the required sample amount.
10:30 Coffee Break
11:00 Combination of Microfluidics and Next-Generation Sequencing for
Targeted Sequencing of Multiple Genomic Loci
Ewen Kirkness, Ph.D., Investigator, J. Craig Venter Institute
The targeted resequencing of discrete genomic loci in human populations is a growing research area where many new methodologies are currently being developed. Previously, targeting has been achieved reliably by PCR, though the procedures applied have been relatively low-throughput and expensive. In order to fully exploit the potential of new sequencing technologies for targeted resequencing, we have employed the RainDance droplet-based technology platform. This generates picoliter-volume PCR reactions, and a sequence enrichment application uses a library of PCR primers in droplets to amplify hundreds of genomic loci in a single tube. We have applied this technology to several human resequencing projects, and compared result to conventional PCR-based approaches.
11:30 FEATURED PRESENTATION
New Approaches in DNA Sequencing
Ronald W. Davis, Ph.D., Professor, Biochemistry & Genetics, Stanford Genome Technology Center
12:00 pm Close of Morning Session
Sponsored by12:15 Luncheon Presentation
Sample Enrichment Strategies for Sequencing with the SOLiD™ System
Michael Rhodes, Ph.D., Product Applications Senior Manager, Genetic Analysis, High Throughput Discovery, Applied Biosystems
Targeted resequencing strategies have been a key focus for translational genomics. There are multiple techniques for accomplishing this and various commercial alternatives. Is there a right one? This discussion will outline the different pathways to enrich samples for use on next generation sequencers, in particular the SOLiD ™ 3 System. The SOLiD ™ 3 System is capable of providing 10X human genome coverage in a single run. At what point is whole genome sequencing a more appropriate approach?"
EXPANDING THE ENVELOPE OF APPLICATIONS
2:00 Chairperson’s Remarks
2:05 Capturing and Sequencing the Protein Coding Genome
Jay Shendure, Ph.D., Assistant Professor, Genome Sciences, University of Washington
Next-generation sequencing technologies have reduced the cost of DNA sequencing by several orders of magnitude. While the routine resequencing of full human genomes continues to be prohibitively expensive for studies involving large numbers of individuals, the cost of resequencing the ~1% of the human genome that is protein-coding, may soon be on par with that of dense genotyping arrays. We are exploring several strategies for massively multiplex capture of discontiguous genomic subsequences, a prerequisite for efficient PCG resequencing. These include an approach based on oligonucleotide libraries derived from programmable microarrays, which then serve as Molecular Inversion Probes, while another approach involves using dense microarrays for capture-by-hybridization. By using HapMap samples and coupling both capture methods to high-throughput sequencing on the Illumina platform, we have observed high sensitivity and specificity for variant discovery at well-covered positions. Ongoing work is primarily directed at optimizing for capture uniformity and efficiency.
2:30 Sequencing Out the Stem Cell Epigenome
R. David Hawkins, Ph.D., Ludwig Institute for Cancer Research, University of California San Diego School of Medicine
We are using ChIP-Seq to identify the location of various histone modifications in the human embryonic stem cell epigenome. We are able to identify distinct patterns, some of which correlate with genomic regulatory elements. An extensive investigation into these patterns should shed light on their role in gene transcription.
2:55 Shading Light into the Composition of Natural Micro- bial Communities using Multiplex Pyrosequencing
Lutz Krause, Ph.D., Bioinformatics, Bioanalytical Science Department, Nestlé Research Center, Switzerland
The recently published multiplex pyrosequencing allows the simultaneous sequencing of hundreds of microbial 16S DNA samples at a low cost. A validation study will be presented, demonstrating that the reconstructed community composition of the analyzed samples strongly depends on the used PCR primers and amplified regions. Furthermore, the power of multiplex pyrosequencing to investigate the taxonomic composition of entire microbial communities is high-lighted on a real-world example.
3:25 Refreshment Break
This session showcases the next-next or “third” generation of sequencing technologies.
4:00 Complete Genomics: Revolutionizing Human Genome SequencingRadoje Drmanac, Ph.D., Chief Scientific Officer, Complete Genomics
Researchers need to compare large numbers of human genomes to be able to characterize rare genetic variants in order to discover the missing links between our genes and diseases. To facilitate that discovery process and provide affordable complete genetic diagnostics, We have developed a new sequencing platform that capitalizes on its advances in both DNA arrays and sequencing assays. Its platform employs the first sub-micron arrays, which are populated with DNA nano-balls™, and uses a non-sequential read technology, referred to as combinatorial probe-anchor ligation or cPAL™, that reduces both reagent consumption and imaging time. Another unique feature of its platform is its long fragment read technology (LFR™) that generates separate sequences for homologous pairs of parental chromosomes. This capability is critical to sequencing diploid human genomes, as it allows heterozygote phasing over large intervals (potentially entire chromosomes), even in areas with high recombination rates. This approach can be used to resolve extensive rearrangements in cancer genomes and determine full-length sequences of alternatively spliced transcripts. We will share its latest genome sequencing data results and provide an update on its technology progress.
4:20 Single Molecule Mega-Genomic Analysis in Nanochannel Array
Michael Boyce-Jacino, Ph.D., President and CEO, BioNanomatrix, Inc.
The BioNanomatrix technology analyzes individual long strands of native genomic DNA in a massively parallel format, avoiding the fragmentation and complex data re-assembly required by other approaches. Highly standardized, semi-conductor fabrication based manufacturing methods promise highly reliable, inexpensive methods for ultra-small sample genome analysis and diagnostics. The affordability, speed and simplicity of the technology are expected to make the routine use of genetic information feasible in broad-ranging applications in sequencing, SVs analysis, molecular diagnostics and personalized medicine.
4:40 The PinPoint Sequencer: High Throughput, Low Cost DNA Sequencing
Jerzy Olejnik, Ph.D., Vice President, Process R&D, Intelligent Bio-Systems, Inc.
Intelligent Bio-Systems’ next-generation sequencing platform is based on proprietary sequencing by synthesis (SBS) chemistry invented at Columbia University. The system utilizes single-stranded, clonally amplified DNA fragments as templates and incorporates specially labeled nucleotide analogs into a growing complementary second strand one base at a time. The instrument design leverages inherently rapid extension and cleavage times that result in higher throughput and lower cost than other currently available systems. The high performance of the PinPoint instrument is achieved by combining fast chemistry cycles, a high speed imaging system and an efficient, ordered-array-based chip.
5:00 Solid-State Nanopores as Single-Molecule Hybridization Detectors for Genomic Sequencing
John Oliver, Ph.D., Vice President Research, NABsys, Inc.
NABsys is developing a new sequencing platform that uses solid-state nanopores as single molecule detectors. Genomic DNA will be randomly cleaved to generate long fragments. The fragments will be hybridized with oligonucleotide probes. The hybridized strands are translocated through a nanopore and the relative positions of the probes are determined. The data for each probe is used to assemble the fragments into probe map. The sequence of the genomic DNA is reconstructed from a collection of probe maps. The method promises to be low cost, and to retain long range information.
5:20 Commercialization of Lightning Terminators™, LaserGen’s Next-Generation Reversible Terminator Chemistry
Michael L. Metzker, Ph.D., President and CEO, LaserGen, Inc.
The demand for DNA sequence information has never been greater, yet current Sanger technology is expensive, time consuming, and labor intensive to meet the ongoing demand. To overcome this challenge, we have developed a novel sequencing platform called cyclic reversible termination (CRT), which differs significantly from Sanger sequencing and pyrosequencing. CRT comprises three steps: incorporation, imaging, and cleavage. At the heart of the CRT method is the reversible terminator (RT). Several groups have created a variety of modified nucleotides, whose function directly affects the performance of the sequencing platform. To enable these nucleotides, a variety of mutations are introduced into the DNA polymerase. This improves incorporation, but at the expense of specificity. We have constructed a microfluidic device that integrates all cycle steps of the CRT method, including reagent delivery and temperature control, UV cleavage, and fluorescence imaging. The advantages of the CRT method are demonstrated by the sequencing of E. coli1655 using a mate-pair, emulsion PCR protocol, the genome of which is useful in providing a benchmark for comparing sequencing metrics such as read-length, accuracy, and coverage with other next-generation platforms.
5:40 Question & Answer Session with Next-Next Gen Speakers
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