Next Generation Sequencing: comparison of the technologies for genome sequencing
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© 2007 GATC Biotech
Next Generation Sequencing: comparison of the technologies for
genome sequencing
DNASTAR Workshop “Deciphering Next Generation Data”
ASHG, San Diego, October 25 2007
Dr. Kerstin Stangier
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
GATC
- Biggest European sequencing service provider with a capacity of150 gigabases / year
- innovative bioinformatic software
- nearly 2 decades of experience and know how
- ISO-certified since 1997
- 100% privately owned, self-financed & independent
- 63 employees in 3 subsidaries & 10 sales offices
- more than 10 000 customers in 40 countries from industry & academia
complete solutions for all sequencing requests:
from DNA preparation to high throughput sequencing of whole genomes and bioinformatics
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Overview
Illumina / Solexa Genetic Analyzer2000 Mb / run
Applied Biosystems ABI 3730XL1 Mb / day
Roche / 454 Genome Sequencer FLX100 Mb / run
Applied BiosystemsSOLiD3000 Mb / run
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Roche / 454 : GS FLX
• Real Time Sequencing by Synthesis
• Chemiluminescence detection in pico titer plates
• Amplification: emulsion PCR
• Pyrosequencing
• up to 400,000 reads / run
• on average 250 bases / read
• up to 100 Mb / run
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Illumina / Solexa: Genetic Analyzer
• Real Time Sequencing by Synthesis
• Clonal Single Molecule Array
• Amplification: bridging PCR
• 60 mio reads / run
• up to 50 bases / read
• 2 Gb / run
• 8 channels, app. 5 mio reads / channel
• Fluorescent labels
• Reversible 3‘OH blocking
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
SOLiD system
• Real Time Sequencing by Ligation
• Emulsion PCR and Beads on slides
• 85 mio reads / run
• Up to 35 bases/read
• 3 Gb / run
• dual fluorescent labels
• 8 individual channels / flowcell
• 2 flowcells / run
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Case study: de novo genome sequencing
Bacterial genome (app. 6.5 MB)
4 x coverage by Sanger sequencing
(plasmid & fosmid clones)
4 x coverage by sequencing with 454 technology (GS 20)
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Case study: de novo genome sequencing
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Case study: hard / full stops
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Case study: hard / full stops
454
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Flowgrams in SeqMan
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Homopolymers
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Case study: de novo genome sequencing
Backbone Sanger sequencing recommended
Sanger sequencing for repeats
454 sequencing for hard stops
Solexa sequencing for homopolymers
For de novo sequencing, a hybrid strategy of
Sanger, 454, and short read sequencing
is highly recommended
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Case study: resequencing of genomes
Bacterial genomes sequenced with
- Genome Analyzer (Illumina / Solexa)
- coverage between 33-fold and 70-fold
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Case study: resequencing of genomes
Bacterial genome (app. 6.5 MB)
- sequencing on a Genome Analyzer (33 bp read length)
3 channels: 13.5 mio reads
- approx. 445 MB raw data
- mapping to “reference“ genome in database: 223 MB
- mapping to sequenced genome (Sanger + 454): 370 MB
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Case study: resequencing of genomes
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Case study: resequencing of genomes
Bacterial genomes: size between 3.3 Mb and 4.6 Mb
- mapped to the reference: between 70% and 92%
- percentage of mapped reads depends on
homology to reference genome
repeats
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Applications GS FLX
• de novo sequencing
• cDNA libraries, ESTs
• amplicon sequencing, long range PCR fragments
• human samples
• BAC pools
• fosmid pools
• metagenomes, biofilm
• transcriptomes
• LAM-PCR
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Applications Genetic Analyzer
• resequencing
• ChIP
• small RNAs
• SAGE, SuperSAGE
• CAGE
• reference sequence is needed!
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© 2007 GATC Biotech
DNASTAR Workshop “Deciphering Next Generation Data” , ASHG, San Diego October 2007 © 2007 GATC Biotech
Applications Genetic Analyzer
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© 2007 GATC Biotech
Thank you
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