the impact of next-generation sequencing technology of genetics elaine r. mardis – 11 february....
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The impact of next-generation sequencing technology of geneticsElaine R. Mardis – 11 February. 2008
Washington School of Medicine, Genome Sequencing Center.
Presented by Jacob Juhn
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“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
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Overview
Next Generation Instruments-Roche (454) GS FLX sequencer-Illumina genome analyzer-Applied Biosystems SOLiD sequencer
Mutation Discover Sequencing clinical isolates in strain-to-
reference sequences Enabling metagenomics Regulatory protein binding
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Overview
Exploring chromatic packaging Future Challenges Concluding Remarks
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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.eduhttp://www.broad.mit.edu/http://www.hgsc.bcm.tmc.edu/http://www.sanger.ac.uk/
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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
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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
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Roche (454) GS FLX sequencer
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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
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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
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Roche (454) GS FLX sequencer
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Roche (454) GS FLX sequencer
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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
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Roche (454) GS FLX sequencer
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Illumina genome analyzer
Introduced in 2006 Concept of ‘sequencing by synthesis’
(SBS) Produce ~32-40bp from tens of millions
of surface amplified DNA fragments
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Illumina genome analyzer
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Illumina genome analyzer
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Illumina genome analyzer
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Illumina genome analyzer
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Illumina genome analyzer
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Illumina genome analyzer
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Illumina genome analyzer
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Illumina genome analyzer
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Illumina genome analyzer
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Illumina genome analyzer
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Applied Biosystems SOLiD sequencer
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Applied Biosystems SOLiD sequencer
Commercial release in October 2007 Unique sequencing catalyzed by DNA
ligase Sequencing by Oligo Ligation and
Detection ~5 days to run / produces 3-4Gb Average read length of 25-35bp
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Applied Biosystems SOLiD sequencer
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Applied Biosystems SOLiD sequencer
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Comparison
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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 Caenorhabditis
elegans
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Clinical Isolates
DNA sequence library from single genomic fragment
Conventional method long process HIV clinical isolate Campylobacter jejuni Mycobacterium tuberculosis
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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
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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
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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
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Future challenges
Human genome / Hap-MAP Little known below phenotype level Re-sequence using next-generation
ChIP-seq / ncRNA increase knowledge of genome variability
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Concluding remarks
Sequence-based genomes relatively young pursuit
Fundamental knowledge being enhanced Time and ingenuity will determine
boundaries