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New High Throughput Sequencing technologies at the Norwegian Sequencing Centre - and beyond
 

New High Throughput Sequencing technologies at the Norwegian Sequencing Centre - and beyond

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A talk I gave at the Microbiology Research Group (University of Oslo) about new High Throughput Sequencing instruments at the Norwegian Sequencing Centre. I also mentioned future upgrades, and the ...

A talk I gave at the Microbiology Research Group (University of Oslo) about new High Throughput Sequencing instruments at the Norwegian Sequencing Centre. I also mentioned future upgrades, and the upcoming nanopore sequencing platform of Oxford nanopore

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    New High Throughput Sequencing technologies at the Norwegian Sequencing Centre - and beyond New High Throughput Sequencing technologies at the Norwegian Sequencing Centre - and beyond Presentation Transcript

    • New High Throughput Sequencing technologies at the Norwegian Sequencing Centre - and beyond Lex Nederbragt, NSC and CEES lex.nederbragt@bio.uio.no
    • Norwegian Sequencing Centre National technology core facility
    • Norwegian Sequencing Centre Offering sequencing servicesGS FLX from Roche/454 HiSeq 2000 from Illumina
    • 1) New instruments:Benchtop sequencers
    • High-throughput sequencing Phase 1: more is better 2005 GS20 200 000 reads 100 bp 0.02 Gb/run 2011 GS FLX+ 1.2 million reads 750 bp 0.7 Gb/run 2006 GA 28 million reads 25 bp 0.7 Gb/run 2011 HiSeq 2000 3 billion reads 2x100 bp 600 Gb/run
    • High-throughput sequencing Phase 2: smaller is better GS Junior from Roche/454 0.04 GB/run 400 bp reads0.7 GB/run700 bp reads MiSeq from Illumina 4.5 GB/run 2x150 bp reads600 GB/run2x100 bp reads PGM from Ion Torrent/ Life Technologies 0.01, 0.1 or 1 GB/run 100 or 200 bp reads
    • High-throughput sequencing Why benchtop sequencing instruments? GS Junior from Roche/454 10 hours23 hours MiSeq from Illumina 27 hours10 days PGM from Ion Torrent/ Life Technologies 3 hours
    • High-throughput sequencing Why benchtop sequencing instruments? Diagnostics Affordable price per instrument Small projects Fast turn around timehttp://pennystockalerts.com/ http://www.highqualitylinkbuildingservice.com/http://www.vetlearn.com/ http://vanillajava.blogspot.com
    • High-throughput sequencingBenchtop sequencing instruments at the NSC ✖ ✔ ✔ ✔ ✔
    • Sequencing technology
    • Sequencing technology Reversible dye terminator sequencing-by-synthesis Reversible terminatorsMetzker 2010 Nat Rev Genet.11(1):31-46
    • Sequencing technology PyrosequencingPPi: pyrophosphate
    • Sequencing technology Free proton current shift sequencing-by-synthesis
    • Error profiles Substitution errors Homopolymer errors
    • Applications Illumina Illumina Platform 454 Ion Torrent HiSeq MiSeqresequencing - +++ ++ +++ de novo +++ + + ++metagenomics +++ ++ + ++ mRNA ++ +++ ++ + miRNA - +++ +++ - ChIP - +++ ++ - DNA meth - +++ + -
    • Announced upgrades HiSeq 2000 HiSeq 2500 2x 250 bp reads 2x150 bp? Rapid run mode 27 hrs 2x150 bp, 90 Gbp 750 bp reads?400 bp reads Proton from Ion Torrent Fall 2012: 10 Gb on Proton I chip, 400 bp 2013: 4 x more wells on Proton II chip
    • Which instrument to choose?
    • Shameless self-promotionflxlexblog.wordpress.com
    • Shameless self-promotion @lexnederbragt
    • 2) New instruments:Pacific Biosciences RS
    • High-throughput sequencing Phase 3: single-moleculeC2 (current) chemistry:Average read length 2500 bp36 000 reads90 MB per ‘run’
    • High-throughput sequencing REVI EW S Technology Pacific Biosciences — Real-time sequencing a Phospholinked hexaphosphate nucleotides G A b 1 nm 00Zero-mode waveguide Limit of detection zone Glass Fluorescence pulse Intensity Epifluorescence detection T Figure 4 | Real-time sequencing. P acific Biosciences’ four-colour r a | The zero-mode waveguide (ZMW) design reduces the observatio fluorescently labelled molecules that enter the detection layer for a the dilemma that DNA polymerases perform optimally when fluore
    • High-throughput sequencingS TechnologyReal-time sequencing Phospholinked hexaphosphate nucleotides G A T C b Limit of detection zone Fluorescence pulse Intensitye detection Time Nature Reviews | Genetics Figure 4 | Real-time sequencing. Pacific Biosciences’ four-colour real-time sequencing method is shown.
    • High-throughput sequencing Library preparationSMRTBelltemplateSMRTBelltemplateStandardSequencingStandardSequencing Generates& pass& each& one& on& molecule& Insert& Large& Sizes& Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& sequenced&CircularConsensusSequencingCircularConsensusSequencing Insert& Small& Sizes& Small& Insert& Sizes& mul8ple& Generates& passes& each& on& molecule& Generates& mul8ple& sequenced& passes& each& on& molecule& sequenced&
    • High-throughput sequencing Read length
    • High-throughput sequencingSMRTBelltemplate Raw reads and subreadsStandardSequencing Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& ‘Subreads’CircularConsensusSequencing Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced&
    • High-throughput sequencingSMRTBelltemplateSMRTBelltemplate Raw read qualityStandardSequencing Generates& pass& each& one& on& molecule& Large& Insert& Sizes&StandardSequencing sequenced& 85-87% accuracyCircularConsensusSequencing Large& Insert& Sizes& Random pass& each& Generates& one& sequenced& errors (!) on& molecule&CircularConsensusSequencing Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced& Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced& 4 to 5 passes: accuracies in the high 90s % 5 passes yields average Q30 (1:1000 chance of error)
    • Applications Illumina Illumina Platform 454 Ion Torrent PacBio HiSeq MiSeq*resequencing - +++ ++ - + de novo +++ + + +++ +++metagenomics +++ ++ + +++ +/- mRNA ++ +++ ++ ++ ++ miRNA - +++ +++ - - ChIP - +++ ++ - - DNA meth - +++ + - !!!SNP validation + - - - ++
    • PacBio and base modifications
    • What use for PacBio?http://www.walker.co.uk/What-Use-Is-a-Moose-9780744578393.aspx
    • PacBio: usesStandardSequencing Short reads  high quality Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced&CircularConsensusSequencing Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced& SNP validation Short tandem repeats!
    • PacBio: usesSMRTBelltemplate Long reads  low qualityStandardSequencing Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& 85-87% accuracyCircularConsensusSequencing Uses? Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced&
    • PacBio: long read uses For de novo For scaffoldinghttp://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf
    • PacBio: long read uses For de novo  Error correct with short readshttp://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf
    • PacBio: long read uses For de novohttp://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf
    • PacBio: long read uses For de novoForthcoming paper by Sergey Koren, Mike Schatz In Nature Biotechnology
    • 3) Preliminary results:Pacific Biosciences RS
    • PacBio: first results Samples Atlantic cod Fish Xhttp://en.wikipedia.org http://www.drawinghowtodraw.com/
    • PacBio: first resultsSMRTBelltemplate LibrariesStandardSequencing Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& 4kb and 17b insert sizesCircularConsensusSequencing Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced&
    • PacBio: first results Raw reads ZMWs Mean readlength30,000 4,00025,000 3,500 3,00020,000 2,50015,000 2,00010,000 1,500 1,000 5,000 500 0 0 cod 4kb cod 17kb Fish X 4kbFish X 4kb Fish X Fish X cod 4kb cod 17kb Fish X 4kb Fish X 4kb Fish X Fish X 17kb 17kb 17kb 17kb Longest read 25,000 20,000 15,000 10,000 5,000 0 cod 4kb cod 17kb Fish X 4kb Fish X 4kb Fish X 17kb Fish X 17kb
    • SMRTBelltemplate PacBio: first resultsLength of longest subread for all raw reads StandardSequencing Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& CircularConsensusSequencing Average length 16,000 Largest Small& Insert& Sizes& 14,000 Generates& mul8ple& passes& each& on& molecule& 12,000 sequenced& 10,000 8,000 6,000 4,000 2,000 0 cod 4kb cod 17kb Fish X 4kb Fish X 4kb Fish X 17kb Fish X 17kb
    • PacBio: first results Length of longest subread for all raw reads Per SMRTCell longest subread length density distribution Fish X Salmon Atlantic Atlantic Cod 4kb libraries Cod 3e−04 17kb libraries 2e−04Density 1e−04 0e+00 0 2000 4000 6000 8000 10000 Maximum subread length
    • PacBio: first resultsMapping to the cod genome Long insert – single pass Short insert – many passes
    • 4) Beyond ‘second generation’ sequencing
    • High-throughput sequencingPhase 1: more is betterPhase 2: smaller is betterPhase 3: single-moleculePhase 4: the sky is the limit?
    • Nanopore sequencing
    • Oxford NanoporeAGBT conference, February 2012
    • Oxford Nanopore AGBT conference, February 2012 100 kbp reads Currently at 4% error 1% error at releaseGridION 2000 nanopores/node tens of Gb data per 24 hour Run until… 20 nodes 1 human genome in 15 minutes
    • Oxford NanoporeAGBT conference, February 2012 MinION 512 nanopores 150mb/hour Up to 6 hours $900
    • Nanopore sequencing Seeing is believinghttp://uwgcm.org
    • Nanopore sequencingDemocratization of sequencing