New High Throughput Sequencing technologies at the Norwegian Sequencing Centre

- and beyond

Lex Nederbragt, NSC and CEESlex.nederbragt@bio.uio.no

Norwegian Sequencing Centre

National technology core facility

Norwegian Sequencing Centre

Offering sequencing services

GS 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 bp0.02 Gb/run

2011 GS FLX+ 1.2 million reads 750 bp0.7 Gb/run

2006 GA 28 million reads 25 bp0.7 Gb/run

2011 HiSeq 2000 3 billion reads 2x100 bp600 Gb/run

High-throughput sequencing

Phase 2: smaller is better

GS Junior from Roche/454

MiSeq from Illumina

PGM from Ion Torrent/Life Technologies

0.04 GB/run400 bp reads

0.7 GB/run700 bp reads

4.5 GB/run2x150 bp reads

600 GB/run2x100 bp reads

0.01, 0.1 or 1 GB/run100 or 200 bp reads

High-throughput sequencing

Why benchtop sequencing instruments?

10 hours

27 hours

23 hours

10 days

3 hours

GS Junior from Roche/454

MiSeq from Illumina

PGM from Ion Torrent/Life Technologies

High-throughput sequencing

Why benchtop sequencing instruments?

Affordable price per instrument Small projects

Diagnostics

Fast turn around time

http://pennystockalerts.com/ http://www.highqualitylinkbuildingservice.com/http://www.vetlearn.com/ http://vanillajava.blogspot.com

High-throughput sequencing

Benchtop sequencing instruments at the NSC

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Sequencing technology

Sequencing technology

Reversible dye terminator sequencing-by-synthesis

Reversible terminators

Metzker 2010 Nat Rev Genet.11(1):31-46

Sequencing technology

Pyrosequencing

PPi: pyrophosphate

Sequencing technology

Free proton current shift sequencing-by-synthesis

Error profiles

Homopolymer errors

Substitution errors

Applications

Platform 454 Illumina HiSeq

Illumina MiSeq Ion Torrent

resequencing - +++ ++ +++de novo +++ + + ++

metagenomics +++ ++ + ++

mRNA ++ +++ ++ +

miRNA - +++ +++ -

ChIP - +++ ++ -

DNA meth - +++ + -

Announced upgrades

750 bp reads?

2x 250 bp reads

Proton from Ion Torrent

Fall 2012: 10 Gb on Proton I chip, 400 bp2013: 4 x more wells on Proton II chip

HiSeq 20002x150 bp?

HiSeq 2500Rapid run mode 27 hrs

2x150 bp, 90 Gbp

400 bp reads

Which instrument to choose?

Shameless self-promotion

flxlexblog.wordpress.com

Shameless self-promotion

@lexnederbragt

2) New instruments:

Pacific Biosciences RS

High-throughput sequencing

Phase 3: single-molecule

C2 (current) chemistry:Average read length 2500 bp36 000 reads90 MB per ‘run’

High-throughput sequencing

Technology

Zero-mode waveguide

High-throughput sequencing

Technology

High-throughput sequencing

Library preparation

High-throughput sequencing

Read length

High-throughput sequencing

Raw reads and subreads

‘Subreads’

High-throughput sequencing

Raw read quality

85-87% accuracyRandom errors (!)

4 to 5 passes: accuracies in the high 90's %5 passes yields average Q30 (1:1000 chance of error)

Applications

Platform 454 Illumina HiSeq

Illumina MiSeq* Ion Torrent PacBio

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: uses

Short reads high quality

SNP validationShort tandem repeats!

PacBio: uses

Long reads low quality

Uses?

85-87% accuracy

PacBio: long read uses

For de novo

http://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf

For scaffolding

PacBio: long read uses

For de novo Error correct with short reads

http://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf

PacBio: long read uses

For de novo

http://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf

PacBio: long read uses

For de novo

Forthcoming paper by Sergey Koren, Mike SchatzIn Nature Biotechnology

3) Preliminary results:

Pacific Biosciences RS

PacBio: first results

Samples

Atlantic cod Fish X

http://www.drawinghowtodraw.com/http://en.wikipedia.org

PacBio: first results

Libraries

4kb and 17b insert sizes

PacBio: first results

Raw reads

cod 4kb cod 17kb Fish X 4kb Fish X 4kb Fish X 17kb

Fish X 17kb

0

5,000

10,000

15,000

20,000

25,000

30,000

ZMWs

cod 4kb cod 17kb Fish X 4kb Fish X 4kb Fish X 17kb

Fish X 17kb

0500

1,0001,5002,0002,5003,0003,5004,000

Mean readlength

cod 4kb cod 17kb Fish X 4kb Fish X 4kb Fish X 17kb Fish X 17kb0

5,000

10,000

15,000

20,000

25,000

Longest read

PacBio: first results

Length of longest subread for all raw reads

PacBio: first results

Length of longest subread for all raw reads

Fish XCod

PacBio: first results

Mapping to the cod genome

Long insert – single pass

Short insert – many passes

4) Beyond ‘second generation’ sequencing

High-throughput sequencing

Phase 1: more is better

Phase 2: smaller is better

Phase 3: single-molecule

Phase 4: the sky is the limit?

Nanopore sequencing

Oxford Nanopore

AGBT conference, February 2012

Oxford Nanopore

AGBT conference, February 2012

100 kbp readsCurrently at 4% error1% error at release

GridION2000 nanopores/nodetens of Gb data per 24 hourRun until…20 nodes 1 human genome in 15 minutes

Oxford Nanopore

AGBT conference, February 2012

MinION

512 nanopores 150mb/hour

Up to 6 hours$900

Nanopore sequencing

Seeing is believing

http://uwgcm.org

Nanopore sequencing

Democratization of sequencing