Aaron J. Mackey, Ph.D.amackey@virginia.edu

Center for Public Health Genomics

Wednesday October 7th, 2009BIMS 853 Special Topics in Cardiovascular Research

source: Francis Ouellette, OICR

“omic” Disease Research

source: Francis Ouellette, OICR

Basics of the “old” technology• Clone the DNA.• Generate a ladder of labeled (colored) molecules that

are different by 1 nucleotide.• Separate mixture on some matrix.• Detect fluorochrome by laser.• Interpret peaks as string of DNA.• Strings are 500 to 1,000 letters long• 1 machine generates 57,000 nucleotides/run• Assemble all strings into a genome.

source: Francis Ouellette, OICR

Basics of the “new” technology• Get DNA.• Attach it to something.• Extend and amplify signal with some color scheme.• Detect fluorochrome by microscopy.• Interpret series of spots as short strings of DNA.• Strings are 30-300 letters long• Multiple images are interpreted as 0.4 to 1.2 GB/run

(1,200,000,000 letters/day). • Map or align strings to one or many genome.

source: Francis Ouellette, OICR

Differences between platforms:

• Nanotechnology used.• Resolution of the image analysis.• Chemistry and enzymology.• Signal to noise detection in the software• Software/images/file size/pipeline• Cost $$$

source: Francis Ouellette, OICR

Genome size: 3000 MbReq'd coverage: 6 12 25

3730 454 FLX Solexabp/read 600 250 32Reads/run 96 400,000 40,000,000 bp/run 57,600 100,000,000 1,280,000,000 #/runs req'd 312,500 360 59

Cost per run 48$ 6,800$ 9,300$ Total cost 15,000,000$ 2,448,000$ 544,922$

Adapted from Richard Wilson, School of Medicine, Washington University, “Sequencing the Cancer Genome” http://tinyurl.com/5f3alk

3 Gb ==

source: Francis Ouellette, OICR

NGS technologies

• Roche/454 Life Sciences• Illumina (Solexa)• ABI SOLiD• Helicos• Complete Genomics• Pacific Biosciences• Polonator

Roche/454 pyrosequencing

454 flowgram

454 has difficulty quantizing luminescence of long homopolymers;problem gets worse with homopolymer length

Roche/454

• first commercially available NGS platform• long reads (most 100-500bp; soon 1000bp)• paired-end module available• relatively expensive runs• homopolymer error rate is high• common uses: metagenomics, bacterial

genome (re)sequencing• James Watson’s genome done entirely on 454• UVA Biology Dept. has one (Martin Wu)

Illumina (Solexa)• 75 bp reads, PE• 150-250 bp fragments• 8 lanes per flowcell• ~3 Gbp per lane• < 5% error rate• available at UVA BRF DNA Core

ABI SOLiD

SOLiD “color space”

ABI SOLiD

• short reads (~35 bp)• cheapest cost/base• high fidelity reads (easy to detect errors)• Common uses: SNP discovery• 1000 genome project• with PET libraries, all applications within

reach …

Comparing Sequencers

Roche (454) Illumina SOLiD

Chemistry Pyrosequencing Polymerase-based Ligation-based

Amplification Emulsion PCR Bridge Amp Emulsion PCR

Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb

Mb/run 100 Mb 1300 Mb 3000 Mb

Time/run 7 h 4 days 5 days

Read length 250 bp 32-40 bp 35 bp

Cost per run (total) $8439 $8950 $17447

Cost per Mb $84.39 $5.97 $5.81

source: Stefan Bekiranov, UVA

Other NGS platforms

• Helicos (Stephen Quake, Stanford)– single molecules on slide– like Illumina, but no PCR, greater density

• Complete Genomics– sequencing factory– 10K human genomes/year, $10K each

• Pacific Biosciences – SMRT– DNA polymerase bound to laser/camera hookup– records a movie of DNA replication with fluoroscent

dNTPs as single strand moves through nanopore• Polonator (Shendure and Church)

– homebrew, $200K flowcell+laser machine– allows custom chemistry protocols

NGS applications

• genome (re)sequencing– de novo genomes: 454 in Bact, small Euks– SNP discovery and genotyping (barcoded pools)– targeted, “deep” gene resequencing– metagenomics

• structural/copy-number variation– Tumor genome SV/CNV: Illumina/PET

• epigenomics – last week’s seminar• RNA-seq: now-generation transcriptomics • ChIP-seq: now-generation DNA-binding

RNA-seq: RNA abundance

RNA-seq: alternative splicing

RNA-seq

• “unbiased” digital measure of abundance– residual PCR artifacts? Helicos says “yes”

• larger dynamic range than microarray– depends on sequencing depth cost

• ability to see alt./edited transcripts– multiple AS sites confounded; 454?

• Total RNA vs. cDNA– 3’ end bias of cDNA– non-polyA transcripts in total RNA

ChIP-seq: protein-DNA binding

PET: Paired End Tag libraries

PET applications

some things I didn’tget to talk about much:

• personal genome sequencing/medicine• microbial metagenomics• ENCODE/modENCODE projects• HapMap project• human 1000 Genome Project (1KGP)• targeted- and/or deep-resequencing• microRNAs, piRNAs, ncRNAs, …• SVs and CNVs (cancer)• read alignment issues (“mapability”)

Questions?

amackey@virginia.edu