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Next Generation Sequencing: comparison of the technologies for genome sequencing

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. GATC. - Biggest European sequencing service provider with a capacity of 150 gigabases / year

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Next Generation Sequencing: comparison of the technologies for genome sequencing

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  1. Next Generation Sequencing: comparison of the technologies for genome sequencing DNASTAR Workshop “Deciphering Next Generation Data” ASHG, San Diego, October 25 2007Dr. Kerstin Stangier

  2. GATC • - Biggest European sequencing service provider with a capacity of 150 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

  3. Overview Applied Biosystems ABI 3730XL 1 Mb / day Roche / 454 Genome Sequencer FLX 100 Mb / run Illumina / Solexa Genetic Analyzer 2000 Mb / run Applied Biosystems SOLiD 3000 Mb / run

  4. 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

  5. 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

  6. 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

  7. 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)

  8. Case study: de novo genome sequencing

  9. Case study: hard / full stops

  10. Case study: hard / full stops 454

  11. Flowgrams in SeqMan

  12. Homopolymers

  13. 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

  14. Case study: resequencing of genomes Bacterial genomes sequenced with - Genome Analyzer (Illumina / Solexa) - coverage between 33-fold and 70-fold

  15. 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

  16. Case study: resequencing of genomes

  17. 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

  18. 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

  19. Applications Genetic Analyzer • resequencing • ChIP • small RNAs • SAGE, SuperSAGE • CAGE • reference sequence is needed!

  20. Applications Genetic Analyzer

  21. Thank youwww.gatc-biotech.com

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