NEXT GENERATION SEQUENCING ON AB SOLID & ION TORRENT PGM - INGER JONASSON UPPSALA GENOME CENTER SCILIFELAB UPPSALA

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NEXT GENERATION SEQUENCING ON AB SOLID & ION TORRENT PGM - INGER JONASSON UPPSALA GENOME CENTER SCILIFELAB UPPSALA
Next Generation Sequencing on
AB SOLiD™ & Ion Torrent PGM™

                   Inger Jonasson
                   Uppsala Genome Center
                   SciLifeLab Uppsala
NEXT GENERATION SEQUENCING ON AB SOLID & ION TORRENT PGM - INGER JONASSON UPPSALA GENOME CENTER SCILIFELAB UPPSALA
Work Flow – Next Generation
            Sequencing
1. Library preparation           2. Emulsion PCR and
                                 bead preparation

  3.Sequencing run       4.Mapping of reads and
                         initial bioinformatic
                         analysis
NEXT GENERATION SEQUENCING ON AB SOLID & ION TORRENT PGM - INGER JONASSON UPPSALA GENOME CENTER SCILIFELAB UPPSALA
SOLiD Library Types
NEXT GENERATION SEQUENCING ON AB SOLID & ION TORRENT PGM - INGER JONASSON UPPSALA GENOME CENTER SCILIFELAB UPPSALA
Fragment Libraries
• Whole Genome re-sequencing
   – SNP/indel detection
• Targeted re-sequencing
   – Exome sequencing or selected region – SNP/indel
     detection
• ChIP-seq
   – Protein binding sites
• Methylation analysis
                                         75 bp          35 bp
• de novo sequencing

                             75 bp single reads or 75 + 35 bp
                             paired end reads. Multiplex options up
                             to 96 barcodes.
NEXT GENERATION SEQUENCING ON AB SOLID & ION TORRENT PGM - INGER JONASSON UPPSALA GENOME CENTER SCILIFELAB UPPSALA
Mate-Pair Libraries

Two tags on the same read with a known tag
distance of 1-10 Kbp.
•   Whole genome re-sequencing
    – SNP/indel detection
    – Identification of structural rearrangements
• de novo sequencing

                           60 bp           60 bp

                      2 x 60 bp reads in the same direction with
                      a known distance between the tags.
NEXT GENERATION SEQUENCING ON AB SOLID & ION TORRENT PGM - INGER JONASSON UPPSALA GENOME CENTER SCILIFELAB UPPSALA
RNA Libraries

 • Whole Transcriptome Analysis.
    Starting material polyA selected or rRNA depleted
    RNA
     –   Gene expression
     –   Splice variants
     –   SNP/indel detection
     –   Novel transcript identification

 • mi-RNA. Starting material size selected total RNA
 WTA 75 bp       35 bp

                           70 + 35 bp paired end reads for Whole
Mi-RNA   35 bp             Transcriptome Analysis. 35 bp single reads for
                           mi-RNA. Multiplex options up to 96.
Overview SOLiD™ Fragment
       Library Design
Workflow –
SOLiD™ Fragment Library I

    10 ng – 20 µg start material

Fragment DNA to 100-200 bp by
sonication with Covaris™ S2 system

End-repair the DNA

Ligate P1 and P2 adaptors to the DNA
Workflow –
  SOLiD™ Fragment Library II

Size-select the DNA on
E-gel® 2% Size Select Gel
or use Agencourt AMPure XP Beads

Nick-translate, then amplify the library

Quantitate the library by quantitative
PCR (qPCR) and Agilent Bioanalyzer
Fragment Library Preparation

           Fragment Library Builder
           Automated fragment library
           preparation, up to 12 samples
           per run.
           Input material; Fragmented DNA
           or RNA.
           Output; Library ready for
           amplification.
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                                            Sequencing read                                        BC read
                                             (35-50 bases)                                         (5 bases)

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                                     P1*                      Target DNA                                     P2*

                                                              5 bases – 20 barcodes

                                                                                 10 bases – 104 barcodes
                                             * P1 & P2 adaptor sequences differ from those in the SOLiDTM kits

                 11                                                                                                       © 2009 Applied Biosystems

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Workflow SOLiD™
Mate-paired Library
Workflow SOLiD™
                    Mate-paired Library I
                 1-3 kb fragment; 5 µg start material
                3-5 kb fragment; 30 µg start material
               5-10 kb fragment; 100 µg start material

Shear the DNA with Hydroshear® or Covaris™ S2 system

Size select the DNA on agarose gel

End-repair the DNA

Ligate MP adaptors to the DNA

Circularize the DNA with internal biotinylated adaptor
Workflow SOLiD™
              Mate-paired Library II
Isolate the circularized DNA with Plasmid Safe™ ATP-
dependent Dnase

Nick-translate the circularized DNA

Digest the DNA with T7 exonuclease and S1 nuclease

Add dA-Tail to the digested DNA

Bind the library to streptavidin beads
Workflow SOLiD™ -
                Mate-paired Library III
Ligate P1-T and P2-T Adaptors to the DNA

Wash the DNA-bound streptavidin beads

Nick-translate and trial-amplify the library

Amplify the library

Size-select and Gel-purify the library

Quantitate the amplified library by performing qPCR and
Agilent Bioanalyzer run.
SOLiD™ Mate-paired Library
        Design
Worklflow -SOLiD™
Whole Transcriptome Analysis Kit I

Startmaterial 1µg rRNA depleted or poly A selected RNA

Fragmentation of RNA with RNase III

Fragmented RNA cleanup

Adaptor hybridization and ligation
 (Adaptor Mix A to sequence 5 end, adaptor Mix B to sequence 3 end)
Workflow -SOLiD™ Whole
  Transcriptome Analysis Kit II

Reverse transcription and RNase H digestion

cDNA library amplification

Amplified library clean-up and size selection
by PAGE or Agencourt AMPure XP beads.

Quantitation of amplified library by qPCR
Workflow - SOLiD™
          Small RNA Expression Kit
Startmaterial; 1-500 ng small (enriched) or total RNA
Adaptor Hybridization / Ligation
 (Adaptor Mix A to sequence 5 end, adaptor Mix B to sequence 3
  end)
Reverse transcription and RNase H digestion

cDNA library amplification

Amplified library clean-up and size selection by PAGE

Quantitate the Small RNA library by qPCR
Quality Control of Libraries
Agilent Bioanalyzer 2100 expert, DNA1000 or
DNA High Sensitivity Chip
Quantitative PCR
Challenges in Library
              Preparation
Quality of starting material DNA or RNA
        - documentation from agarose gel or Agilent
Bioanalyzer

Variation in size after fragmentation due to type of starting
material
        - Hydroshear – shorter fragments than expected
        - Sonication – longer fragments than expected

Variation in measurement of DNA concentration
        - Nanodrop, Bioanalyzer or “Qubit”
”Next Generation Sequencing”
             Work Flow
1. Library preparation           2. Emulsion PCR and
                                 bead preparation

  3.Sequencing run       4.Mapping of reads and
                         initial bioinformatic
                         analysis
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             • Prepare the emulsion PCR (ePCR) reaction.                                                    not be

                  (Oil phase, water phase, SOLiD™ P1 DNA beads).

ar,          • Perform the emulsion break and wash.

             • Enrich for the templated beads.

             • Modify the 3’ ends.

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6 lanes per flow chip
170M beads per lane.
100-120M beads usually generates high quality reads.
”Next Generation Sequencing”
             Work Flow
1. Library preparation           2. Emulsion PCR and
                                 bead preparation

  3.Sequencing run       4.Mapping of reads and
                         initial bioinformatic
                         analysis
SOLiD 5500XL
5500xl SOLiD™ Sequencer
                                                                  5500xl SOLiD™ Sequencer
                                                                                                       30‐45 Gb/day
       Gb/day      [1]                                   20‐30 Gb/day
                                                                                                       (Nano‐beads)
                                                          2 Genomes                                    >3 Genomes
       Samples/run [2]                                     24 Exomes                                    40 Exomes
                                                       12 Transcriptomes                            20 Transcriptomes

       System Accuracy [3]                                                     Up to 99.99%
                                                                               MP: 60x60bp
       Read Length                                                              PE: 75x35bp
                                                                              Fragment: 75bp

       Independent lanes                                                           1 to 12

                                                                             96 for RNA, DNA
       Multiplexing
                                                                       7 days for 60x60bp (12 lanes)
       Run Time                                                         7 days for 75x35bp (12 lanes)
        For Research Use Only.
                                                                            1 day for 35bp (1 lane)
                                     Not intended for any animal or human therapeutic or diagnostic use
       [1] Gb/day range represents minimum to typical expected performance; nano-bead performance projected using 470k
       nano-beads per image panel
       [2] ~30x coverage for human genome; 100x-200x coverage for Exomes; Whole Transcriptome ≥100 M reads/sample
       [3] Reference-free base sequence when using ECC module; ECC requires additional run time and sequencing
       chemistry
38 | Life Technologies Proprietary | 2/2/12
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             •I

                                                                Will be presented in
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                                                                next lecture…….

                 39                                                               © 2008 Applied Biosystems

 Slide number:        Presentation date/confidentiality line:                           Copyright: 10 pt.
”Next Generation Sequencing”
             Work Flow
1. Library preparation           2. Emulsion PCR and
                                 bead preparation

  3.Sequencing run       4.Mapping of reads and
                         initial bioinformatic
                         analysis
Bioinformatics
Platform analysis
• Mapping to reference
• Standard analysis pipelines
  –   SNP detection
  –   Pairing and inversion pipeline
  –   Whole transcriptome
  –   Small RNA
• Data transfer to Uppnex

 More bioinformatics in
 next lecture…….
SOLiD projects & samples

                   Fragment
                                 RNA     Mate-Pair    Seq
        Projects   Libraries
                               Libraries Libraries   Slides

2008      14                                          25

2009      37          90          41        45        53

2010      36         118         109        25        42

2011      56         451          33         6        87
SOLiD Projects
Ion Torrent status

From November 2011
• 7 projects including 20 samples
• 100 bp or 200 bp single reads
• 314 and 316 chip

Sequencing Chemistry and Analysis of data
in next lecture……….
Ion Torrent status
More optimization and evaluation
of protocols to be done
• Fragmentation
   – Covaris vs enzymatic
• Tight size fraction critical, resolution and
  reproducibility important
   – E-gels vs Pippin prep or Caliper

• Emulsion PCR; Yield and
  Quality of templated spheres
   – Manual ePCR vs IonTouch
Frequently Asked Questions

How much start material is needed?

10 ng – 20 µg DNA for Fragment Library.

At least 5 µg DNA for Mate-paired Library.

1-500 ng RNA for mi-RNA Small Expression
RNA Analysis.

1 µg rRNA depleted or polyA selected RNA for
Whole Transcriptome Analysis.
Uppsala Genome Center
Platform director
Ulf Gyllensten

Platform manager
Inger Jonasson

Bioinformatics
Adam Ameur
Ignas Bunikis
Christian Tellgren-
Roth

Technical staff
Michela Asp
Ulrika Broström
Ida Höijer
Susana Häggqvist
Nina Lager
Cecilia Lindau
Anne-Christine
Lindström
Linnea Nyberg

                      Ida Höijer, Ulf Gyllensten, Cecilia Lindau, Linnea Nyberg, Michaela Asp,
www.igp.uu.se
www.scilifelab.se
                      Christian Tellgren-Roth, Nina Lager, Ulrika Broström, Susana Häggqvist,
                      Anne-Christine Lindström, Inger Jonasson, Adam Ameur (missing; Ignas Bunikis)
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