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
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
SOLiD Library Types
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.
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.
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 DesignWorkflow –
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 DNAWorkflow – 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.Template release: Date Indications in white = Locked elements
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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
Slide number: Presentation date/confidentiality line: Copyright: 10 pt.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 adaptorWorkflow 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 beadsWorkflow 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
DesignWorklflow -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 qPCRQuality 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
analysisTemplate release: Date Indications in white = Locked elements
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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.
24 © 2008 Applied Biosystems
Slide number: Presentation date/confidentiality line: Copyright: 10 pt.Template release: Date Indications in white = Locked elements
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26 © 2008 Applied Biosystems
Slide number: Presentation date/confidentiality line: Copyright: 10 pt.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
analysisSOLiD 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 | 2/2/12Template release: Date Indications in white = Locked elements
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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
analysisBioinformatics 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 87SOLiD 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)You can also read
