The impact of next-generation sequencing technology of genetics Elaine R. Mardis – 11 February....

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

“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 Biosystems SOLiD 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.eduhttp://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

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

Roche (454) GS FLX 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

Roche (454) GS FLX sequencer

Illumina genome analyzer

Introduced in 2006 Concept of ‘sequencing by synthesis’

(SBS) Produce ~32-40bp from tens of millions

of surface amplified DNA fragments

Illumina genome analyzer

Illumina genome analyzer

Illumina genome analyzer

Illumina genome analyzer

Illumina genome analyzer

Illumina genome analyzer

Illumina genome analyzer

Illumina genome analyzer

Illumina genome analyzer

Illumina genome analyzer

Applied Biosystems SOLiD sequencer

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

Applied Biosystems SOLiD sequencer

Applied Biosystems SOLiD sequencer

Comparison

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

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