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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 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 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 reads
0.7 GB/run
700 bp reads


                                    MiSeq from Illumina
                                          4.5 GB/run
                                        2x150 bp reads
600 GB/run
2x100 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 hours

23 hours



                            MiSeq from Illumina
                                  27 hours

10 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 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

                                 ✖
 ✔
 ✔                               ✔
                                 ✔
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



        Substitution errors



        Homopolymer errors
Applications




                         Illumina   Illumina
     Platform   454                            Ion Torrent
                           HiSeq     MiSeq
resequencing     -         +++        ++          +++
  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-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 bp
36 000 reads
90 MB per ‘run’
High-throughput sequencing
                      REVI EW S
                          Technology
                      Pacific Biosciences — Real-time sequencing
                      a                                                        Phospholinked hexaphosphate nucleotides

                                                                                                 G                       A

                                                                   b




                      1 nm
                      00
Zero-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 sequencing
S
                                                                        Technology
Real-time sequencing
                                   Phospholinked hexaphosphate nucleotides

                                                     G                       A               T                      C

                       b




                                            Limit of detection zone



                                                   Fluorescence pulse
                       Intensity




e detection                                                                      Time
                                                                                                  Nature Reviews | Genetics
        Figure 4 | Real-time sequencing. Pacific Biosciences’ four-colour real-time sequencing method is shown.
High-throughput sequencing

                          Library preparation

SMRTBell'template'
SMRTBell'template'




Standard'Sequencing'
Standard'Sequencing'

                                          Generates& pass& each&
                                                   one&  on&   molecule&
           Insert&
      Large&     Sizes&                   Generates& pass& each&
                                                   one&  on&   molecule&
      Large&
           Insert&
                 Sizes&                   sequenced&
                                          sequenced&


Circular'Consensus'Sequencing'
Circular'Consensus'Sequencing'


         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 sequencing
SMRTBell'template'


                          Raw reads and subreads

Standard'Sequencing'


                                           Generates& pass& each&
                                                    one&  on&   molecule&
      Large&
           Insert&
                 Sizes&
                                           sequenced&


                                          ‘Subreads’
Circular'Consensus'Sequencing'


    Small&
         Insert&
               Sizes&


                                           Generates&
                                                    mul8ple&
                                                           passes& each&
                                                                 on&   molecule&
                                           sequenced&
High-throughput sequencing
SMRTBell'template'


SMRTBell'template'
                                Raw read quality

Standard'Sequencing'


                                                       Generates& pass& each&
                                                                one&  on&   molecule&
     Large&
          Insert&
                Sizes&
Standard'Sequencing'                                   sequenced&


                                                       85-87% accuracy
Circular'Consensus'Sequencing'
      Large&
           Insert&
                 Sizes&                                Random pass& each&
                                                       Generates&
                                                                one&
                                                       sequenced&
                                                                    errors (!)
                                                                      on&   molecule&




Circular'Consensus'Sequencing'
     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 90's %
                          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: uses
Standard'Sequencing'
                        Short reads  high quality
                                           Generates& pass& each&
                                                    one&  on&   molecule&
      Large&
           Insert&
                 Sizes&
                                           sequenced&



Circular'Consensus'Sequencing'


    Small&
         Insert&
               Sizes&


                                           Generates&
                                                    mul8ple&
                                                           passes& each&
                                                                 on&   molecule&
                                           sequenced&




                             SNP validation
                          Short tandem repeats!
PacBio: uses
SMRTBell'template'


                          Long reads  low quality

Standard'Sequencing'


                                            Generates& pass& each&
                                                     one&  on&   molecule&
      Large&
           Insert&
                 Sizes&
                                            sequenced&


                                            85-87% accuracy
Circular'Consensus'Sequencing'

                                   Uses?
    Small&
         Insert&
               Sizes&


                                            Generates&
                                                     mul8ple&
                                                            passes& each&
                                                                  on&   molecule&
                                            sequenced&
PacBio: long read uses

                  For de novo




               For scaffolding




http://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf
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 Schatz
           In Nature Biotechnology
3) Preliminary results:

Pacific Biosciences RS
PacBio: first results

                          Samples




     Atlantic cod                             Fish X




http://en.wikipedia.org          http://www.drawinghowtodraw.com/
PacBio: first results
SMRTBell'template'


                                 Libraries

Standard'Sequencing'


                                             Generates& pass& each&
                                                      one&  on&   molecule&
      Large&
           Insert&
                 Sizes&
                                             sequenced&


 4kb and 17b insert sizes
Circular'Consensus'Sequencing'


    Small&
         Insert&
               Sizes&


                                             Generates&
                                                      mul8ple&
                                                             passes& each&
                                                                   on&   molecule&
                                             sequenced&
PacBio: first results

                                                            Raw reads
                          ZMWs                                                                Mean readlength
30,000                                                                   4,000
25,000                                                                   3,500
                                                                         3,000
20,000
                                                                         2,500
15,000                                                                   2,000
10,000                                                                   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
SMRTBell'template'
                            PacBio: first results

Length of longest subread for all raw reads
  Standard'Sequencing'


                                                                   Generates& pass& each&
                                                                            one&  on&   molecule&
       Large&
            Insert&
                  Sizes&
                                                                   sequenced&



 Circular'Consensus'Sequencing'
                                                    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−04
Density

          1e−04
          0e+00




                    0            2000                4000              6000       8000        10000

                                                         Maximum subread length
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 reads
                             Currently at 4% error
                             1% error at release




GridION
                  2000 nanopores/node
                  tens of Gb data per 24 hour
                  Run 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

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

  • 1. New High Throughput Sequencing technologies at the Norwegian Sequencing Centre - and beyond Lex Nederbragt, NSC and CEES lex.nederbragt@bio.uio.no
  • 2. Norwegian Sequencing Centre National technology core facility
  • 3. Norwegian Sequencing Centre Offering sequencing services GS FLX from Roche/454 HiSeq 2000 from Illumina
  • 5. 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
  • 6. High-throughput sequencing Phase 2: smaller is better GS Junior from Roche/454 0.04 GB/run 400 bp reads 0.7 GB/run 700 bp reads MiSeq from Illumina 4.5 GB/run 2x150 bp reads 600 GB/run 2x100 bp reads PGM from Ion Torrent/ Life Technologies 0.01, 0.1 or 1 GB/run 100 or 200 bp reads
  • 7. High-throughput sequencing Why benchtop sequencing instruments? GS Junior from Roche/454 10 hours 23 hours MiSeq from Illumina 27 hours 10 days PGM from Ion Torrent/ Life Technologies 3 hours
  • 8. High-throughput sequencing Why benchtop sequencing instruments? Diagnostics Affordable price per instrument Small projects Fast turn around time http://pennystockalerts.com/ http://www.highqualitylinkbuildingservice.com/ http://www.vetlearn.com/ http://vanillajava.blogspot.com
  • 9. High-throughput sequencing Benchtop sequencing instruments at the NSC ✖ ✔ ✔ ✔ ✔
  • 11. Sequencing technology Reversible dye terminator sequencing-by-synthesis Reversible terminators Metzker 2010 Nat Rev Genet.11(1):31-46
  • 12. Sequencing technology Pyrosequencing PPi: pyrophosphate
  • 13. Sequencing technology Free proton current shift sequencing-by-synthesis
  • 14. Error profiles Substitution errors Homopolymer errors
  • 15. Applications Illumina Illumina Platform 454 Ion Torrent HiSeq MiSeq resequencing - +++ ++ +++ de novo +++ + + ++ metagenomics +++ ++ + ++ mRNA ++ +++ ++ + miRNA - +++ +++ - ChIP - +++ ++ - DNA meth - +++ + -
  • 16. 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
  • 19. Shameless self-promotion @lexnederbragt
  • 20. 2) New instruments: Pacific Biosciences RS
  • 21. High-throughput sequencing Phase 3: single-molecule C2 (current) chemistry: Average read length 2500 bp 36 000 reads 90 MB per ‘run’
  • 22. High-throughput sequencing REVI EW S Technology Pacific Biosciences — Real-time sequencing a Phospholinked hexaphosphate nucleotides G A b 1 nm 00 Zero-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
  • 23. High-throughput sequencing S Technology Real-time sequencing Phospholinked hexaphosphate nucleotides G A T C b Limit of detection zone Fluorescence pulse Intensity e detection Time Nature Reviews | Genetics Figure 4 | Real-time sequencing. Pacific Biosciences’ four-colour real-time sequencing method is shown.
  • 24. High-throughput sequencing Library preparation SMRTBell'template' SMRTBell'template' Standard'Sequencing' Standard'Sequencing' Generates& pass& each& one& on& molecule& Insert& Large& Sizes& Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& sequenced& Circular'Consensus'Sequencing' Circular'Consensus'Sequencing' Insert& Small& Sizes& Small& Insert& Sizes& mul8ple& Generates& passes& each& on& molecule& Generates& mul8ple& sequenced& passes& each& on& molecule& sequenced&
  • 26. High-throughput sequencing SMRTBell'template' Raw reads and subreads Standard'Sequencing' Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& ‘Subreads’ Circular'Consensus'Sequencing' Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced&
  • 27. High-throughput sequencing SMRTBell'template' SMRTBell'template' Raw read quality Standard'Sequencing' Generates& pass& each& one& on& molecule& Large& Insert& Sizes& Standard'Sequencing' sequenced& 85-87% accuracy Circular'Consensus'Sequencing' Large& Insert& Sizes& Random pass& each& Generates& one& sequenced& errors (!) on& molecule& Circular'Consensus'Sequencing' 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 90's % 5 passes yields average Q30 (1:1000 chance of error)
  • 28. Applications Illumina Illumina Platform 454 Ion Torrent PacBio HiSeq MiSeq* resequencing - +++ ++ - + de novo +++ + + +++ +++ metagenomics +++ ++ + +++ +/- mRNA ++ +++ ++ ++ ++ miRNA - +++ +++ - - ChIP - +++ ++ - - DNA meth - +++ + - !!! SNP validation + - - - ++
  • 29. PacBio and base modifications
  • 30. What use for PacBio? http://www.walker.co.uk/What-Use-Is-a-Moose-9780744578393.aspx
  • 31. PacBio: uses Standard'Sequencing' Short reads  high quality Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& Circular'Consensus'Sequencing' Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced& SNP validation Short tandem repeats!
  • 32. PacBio: uses SMRTBell'template' Long reads  low quality Standard'Sequencing' Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& 85-87% accuracy Circular'Consensus'Sequencing' Uses? Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced&
  • 33. PacBio: long read uses For de novo For scaffolding http://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf
  • 34. PacBio: long read uses For de novo  Error correct with short reads http://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf
  • 35. PacBio: long read uses For de novo http://schatzlab.cshl.edu/presentations/2012-01-17.PAG.SMRTassembly.pdf
  • 36. PacBio: long read uses For de novo Forthcoming paper by Sergey Koren, Mike Schatz In Nature Biotechnology
  • 38. PacBio: first results Samples Atlantic cod Fish X http://en.wikipedia.org http://www.drawinghowtodraw.com/
  • 39. PacBio: first results SMRTBell'template' Libraries Standard'Sequencing' Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& 4kb and 17b insert sizes Circular'Consensus'Sequencing' Small& Insert& Sizes& Generates& mul8ple& passes& each& on& molecule& sequenced&
  • 40. PacBio: first results Raw reads ZMWs Mean readlength 30,000 4,000 25,000 3,500 3,000 20,000 2,500 15,000 2,000 10,000 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
  • 41. SMRTBell'template' PacBio: first results Length of longest subread for all raw reads Standard'Sequencing' Generates& pass& each& one& on& molecule& Large& Insert& Sizes& sequenced& Circular'Consensus'Sequencing' 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
  • 42. 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−04 Density 1e−04 0e+00 0 2000 4000 6000 8000 10000 Maximum subread length
  • 43. PacBio: first results Mapping to the cod genome Long insert – single pass Short insert – many passes
  • 44. 4) Beyond ‘second generation’ sequencing
  • 45. High-throughput sequencing Phase 1: more is better Phase 2: smaller is better Phase 3: single-molecule Phase 4: the sky is the limit?
  • 48. Oxford Nanopore AGBT conference, February 2012 100 kbp reads Currently at 4% error 1% error at release GridION 2000 nanopores/node tens of Gb data per 24 hour Run until… 20 nodes 1 human genome in 15 minutes
  • 49. Oxford Nanopore AGBT conference, February 2012 MinION 512 nanopores 150mb/hour Up to 6 hours $900
  • 50. Nanopore sequencing Seeing is believing http://uwgcm.org