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The impact of next-generation sequencing technology of genetics. Elaine R. Mardis – 11 February . 2008 Washington School of Medicine, Genome Sequencing Center. Presented by Jacob Juhn.
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The impact of next-generation sequencing technology of genetics Elaine R. Mardis – 11 February. 2008 Washington School of Medicine, Genome Sequencing Center. Presented by Jacob Juhn
“If one accepts that the fundamental pursuit of genetics is to determine the genotypes that explain phenotypes, the meteoric increase of DNA sequence information applied toward that pursuit has nowhere to go but up.” -Elaine R. Mardis
Overview • Next Generation Instruments -Roche (454) GS FLX sequencer-Illumina genome analyzer-Applied BiosystemsSOLiD sequencer • Mutation Discover • Sequencing clinical isolates in strain-to-reference sequences • Enabling metagenomics • Regulatory protein binding
Overview • Exploring chromatic packaging • Future Challenges • Concluding Remarks
Preface • Dideoxynucloetide sequencing of DNA major changes • Cost per reaction of DNA sequencing Fallen / Moore’s Law (Especially over last 5 years) • High-throughput DNA sequencing performed by “handful” of sites : http://genome.wustl.edu http://www.broad.mit.edu/ http://www.hgsc.bcm.tmc.edu/ http://www.sanger.ac.uk/
Preface – next generation instruments • New sequencing instruments revolutionizing genetics. • Process millions of sequence reads in parallel rather than 96 at a time. • Fragment libraries not subject to vector-based cloning and Escherichia coli-based amplification stages • The workflow to produce next-generation sequence-ready libraries is straight foward
Preface – next generation instruments • Relatively little input DNA needed for library • Produce shorter read lengths (*35-250bp) compared to capillary sequencers (650-800bp) • Accuracy of their sequencings and quality values not understood • Labs underway to benchmark relative to capillary electrophoresis *Depending on platform
Roche (454) GS FLX sequencer • Introduced in 2004 • ‘Pyrosequencing’ – pyrophosphate molecule released on nucleotide incorporation by DNA polymerase • Reactions produce light from cleavage of oxyluciferin by luciferase • DNA strands amplified en masse by emulsion PCR
Roche (454) GS FLX sequencer • Emulsion PCR use mixed oil/aqueos mixture to isolate agarose beads • Has unique DNA fragment, aqueous micelles contain PCR reactants • Pipetting micelles in microtiter plate / performing temperature cycle, >1,000,000 sequence 454 beads produced in matter of hours! • Several thousands added to 454 picotiter plate • Picotiter plate placed in genomic sequencer
Roche (454) GS FLX sequencer • Single nucleotide pattern match sequences of four nucleotide, enables 454 software calibrate light emitted. • Signals recorded during the run for each reporting bead position on PTP are translated into a sequence • Several quality-checking steps remove poor quality sequences
Illumina genome analyzer • Introduced in 2006 • Concept of ‘sequencing by synthesis’ (SBS) • Produce ~32-40bp from tens of millions of surface amplified DNA fragments
Applied BiosystemsSOLiD sequencer • Commercial release in October 2007 • Unique sequencing catalyzed by DNA ligase • Sequencing by OligoLigation and Detection • ~5 days to run / produces 3-4Gb • Average read length of 25-35bp
Mutation Discovery • Old ways used PCR to amplify genomic regions • Roche sequencer detect rare variants / alleviate noisy capillary sequence data • 10,000 human exons using primers / parallel approach • Significantly faster and less expensive • Single Illumina run found Caenorhabditiselegans
Clinical Isolates • DNA sequence library from single genomic fragment • Conventional method long process • HIV clinical isolate • Campylobacter jejuni • Mycobacterium tuberculosis
Enabling metagenomics • Sequencing DNA from uncultured, unpurified microbial and/or viral population • “Who’s there?” • Cost too high with conventional capillary platforms • Symbiotic microbes • ‘human microbiome’ characterize with next-generation sequencing • Roche used in process
Regulatory protein binding • Chromatin immunoprecipitation (ChIP) • Old method replaced by next-generation • Both methods complementary in application • ChIP likely to contribute significantly to how protein binding sites are regulated
Exploring chromatin packaging • How genomic DNA packaged into histones • 454-based study for C. elegans genome • ChIP-seq w/Solexa technology • Combining techniques to further explore possibilties
Future challenges • Human genome / Hap-MAP • Little known below phenotype level • Re-sequence using next-generation • ChIP-seq / ncRNA increase knowledge of genome variability
Concluding remarks • Sequence-based genomes relatively young pursuit • Fundamental knowledge being enhanced • Time and ingenuity will determine boundaries
