Next Generation Sequencers

Transcription

1 Next Generation Sequencers

2 Outline Quick Review - Fluorescent DNA sequencing (Sanger sequencing) throughput Next or 2nd generation sequencing methods Enabling technologies for library preparation Sequencing methods Costs Where to obtain 2 nd generation sequencing services.

3 The previous standard for DNA The ABI 3700 capillary electrophoresis sequencer Sanger sequencing 4-colors (dye terminator or dye primer 96 lanes 4-6 hour run 750 bp reads 72,000 bp/run 18,000 bp/hour $2-10/sample depending on scale sequencing

4 Shotgun sequencing Genome of interest Bacterial genome Fragment (nebulize, french press, Sonicate) and Subclone (m13, plasmid) Sequence and assemble.gtctacctgtactgatctagc.... CCTGTACTGATCTAGCATTA.... GTACTGATCTAGCATTACG... Finishing Assume: 4Mbp genome,8x coverage, 750bp reads yields approx 43,000 reads $2/read -> $86k not including library production costs

5 Library production for Sanger sequencing Process Obtain DNA (inexpensive). Fragment and size select DNA (inexpensive). Repair ends, ligate on adapters and clone into vectors and transform into bacteria (inexpensive). Plate on solid media, pick several 1000 clones and grow them (moderately expensive). Isolate DNA from several 1000 clones for sequencing (expensive). Issues It wasn t unusual to spend $0.50-1/clone sequenced. Cloning introduces bias as some sequences don t clone well in some bacteria (toxic to the bacteria, unusual GC content, etc).

6 Next-gen sequencing: Enabling technologies Library construction via massively parallel PCR methods (no cloning) Emulsion based PCR (empcr) PCR colonies (polonies) Alternate sequencing technologies Pyro sequencing (Roche/454) and similar method (Life Technologies/Ion Torrent). Reversible dye terminators (Solexa) Ligation based sequencing (ABI)

7 Library Preparation Bacterial genome Fragment, size select Ligate adapters B B A B B A B A B B A A A B B A

8 Library Preparation (2) B B B B A A B B A A A A A A = biotin Capture with streptavidin Denature B released A

9 Emulsion Based Clonal Amplification A + PCR Reagents B Adapter carrying library DNA Mix DNA Library & capture beads (limited dilution) + Emulsion Oil Create Water-in-oil emulsion Micro-reactors Break micro-reactors Isolate DNA containing beads Perform emulsion PCR Generation of millions of clonally amplified sequencing templates on each bead Millions of beads in a single tube or 96 well plate No cloning and colony picking

10 Polonies (PCR colonies - used by Solexa)

11 Polonies (continued)

12 Next gen sequencing methods Sequencing by synthesis Pyrosequencing - used by 454 (now purchased by Roche) IonTorrent/Life Technologies Reversible dye terminators - used by Solexa (now purchased by Illumina) Sequencing by ligation - used by Applied Biosystems.

13 DNA Replication single stranded DNA binding proteins helicase primosome primase 3 5 replicating 3 5 DNA polymerase III active sites 3 5 RNA primer 3 DNA polymerase I ligase 5

14 The 3 hydroxyl group is the point of attachment of the next base 454 detects this + P P H+ Ion Torrent detects this

15 Image from Biotage.com Pyrosequencing

16 Pyrosequencing Originally done in 96 well plates by affixing the DNA to the surface. Commercialized by Pyrosequencing AB in Sweden Biotage Qiagen. Currently available from Qiagen in their PyroMark Q24 and Q96 instruments. Typical application is rapid genotyping (by sequencing) of 24 or 96 samples in parallel. Read lengths are bp but typically used to sequence a few variant bases down stream of a primer.

17 Bead Loading on PicoTiterPlate TM The µm DNA beads and enzyme carrying beads are loaded into the wells of a PicoTiterPlate TM by centrifugal force. In a typical sequencing run 25-40% of available PicoTiterPlate TM wells are loaded with DNA beads. PicoTiterPlateTM Fused optical fibers with etched wells Dimensions: 70x75 mm Number of wells: 1.6 million Pyrosequencing is performed in the plates.

18 Current characteristics of the GS-FLX sequencer (454/Roche) 1.2M reads 7 or 23 hour run, 2-3 days of prep time Average read length 450bp or 700bp Cost/run = $8000 Total bp = 700Mbp Net - 87 kbp/$1

19 Ion Torrent technology Similar to 454 in concept except the hydrogen ion is detected (instead of the Ppi) Ion Torrent uses a high-density array of micromachined wells in a semiconductor chip to serve a miniature ph meters. Each well holds a different DNA template generated by emulsion PCR. Beneath the wells is an ion-sensitive layer and beneath that a proprietary Ion sensor.

20 Ion Torrent

21 Current characteristics of the # of sensors Ion Torrent PGM 1.2 M 6.2 M 11.1M Cost per run $500-$1000 or around 400k-1M bp/$

22 Ion Torrent s Ion Proton Sequencer Scheduled for release in Q3 of 2012 $149,000 instrument 165M sensors at launch and 660M sensors in 2013 Claim is that this instrument will deliver a full human genome sequence (20x+) for about $1000/genome

23 Illumina Next Gen Sequencing Method Reversible dye terminator chemistry

24

25 Current characteristics of the Solexa 1G sequencer >150,000,000 reads 3-7 day run time, 2-3 days of prep time Average read length 75bp ( bp possible with some protocols) Cost/run = $3000 Total bp = 5Gbp Net 1.5Mbp/$1

26 Sequencing by ligation (ABI-Solid)

27 Current characteristics of the ABI Solid sequencer 800,000,000 reads 2-6 day run time, 3-4 days of prep time Average read length 2x 60bp (mate paired), 75bp for one fragment Cost/run = $8000 Total bp = 60 Gbp Net 7.5Mbp/$1

28 2 nd Generation Sequencing technologies - compared Instrument Technology Reads/run Read length (bp) 454 (Roche) empcr, pyro sequencing IonTorrent (Life Technologies) Solexa (Illumina) Solid (ABI) empcr, H+ detection Polonies, cleavable dye terminators empcr, ligation with cleavable dye terminators Cost/run Bp/$ 1,200, $ kbp 11,000, $1000 1M 150,000, $ Mbp 800,000, $ Mbp

29 Pluses and minuses of current Next gen sequencers Pyrosequencing/Ion Torrent (-) difficult to resolve long homopolymer repeats - e.g. it s hard to distinguish A 7 from A 8 (only 12% more light for A 8 relative to A 7 ). (+) Long reads possible bp now Solid and Solexa systems - Reversible dye terminators or cleavable dye labeled oligos (-) Short reads due to repetitive yield of the chemistry (+) Higher density now Less expensive per base pair

30 Opportunities created by next gen sequencing technology Expression analysis Sequencing is replacing microarrays as the best/least expensive way to measure gene expression at the RNA level Identification of genetic variation The ability to sequence many individuals at low cost allows the detection of rare alleles. Large scale sequencing of clinical isolates of pathogens to correlate genetic variation with virulence Diagnostic sequencing At $1000/genome, sequencing is less expensive than a typical pre-natal test. If we split the cost of a run across many samples, sequencing is the cheapest way to ID pathogens.

31 First generation sequencers Second generation sequ

32 The future of DNA sequencing In the next 5 years, the cost of DNA sequencing will become almost negligible. A cost of $1000 for a human genome is less than the current cost of a prenatal test. Sequencing is rapidly replacing other technologies (microarrays) for measuring relative levels of mrna s or for doing other nucleic acid assays (e.g. measurement of TF binding sites, etc.).

33 Assays that have been demonstrated using Next Gen sequencing Image from: Sequence census methods for functional genomics Barbara Wold & Richard M Myers, Nature Methods - 5, (2008)

34 RNA-Seq From: Mapping and quantifying mammalian transcriptomes by RNA- Seq Ali Mortazavi et.al, Nat. Methods vol 5:207, (2008). Isolation of mrna, hydrolysis to randomly shear RNA, RT with random primers to make cdna, sequencing, mapping sequences back to genome, count to yield RPKM (reads per kilobase of exon model per million mapped reads)

35 Bottom line Sequencing is fast and cheap now. Current costs to sequence a bacterial genome $500-$2000 Current costs to sequence a human genome $5000-$10,000 All microarray applications are being replaced by sequencing. Data analysis, data shipping and data storage will soon be more expensive than data production. Personal genome sequencing is coming to a diagnostic lab near you within the next 2-5 years.

36 Where can I access 2 nd generation sequencing technologies? Visit Put in the word sequencing University of Washington - CEEH Facility Core #1: Functional Genomics & Proteomics (454, Illumina and soon an Ion Torrent), High Throughput Genomics Unit (Illumina) Fred Hutchison Cancer Researc Center Genomics Resource at the Fred Hutchinson Cancer Research Center (Illumina and 454) University of Idaho IBEST DNA Sequence Analysis Core Facility (454) Institute for Systems Biology - DNA Sequencing Core (Illumina) Most companies also have service providers or provide service in-house