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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

Next Generation Sequencing: comparison of the technologies for genome sequencing

DNASTAR Workshop “Deciphering Next Generation Data”

ASHG, San Diego, October 25 2007Dr. Kerstin Stangier

slide2
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
overview
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

roche 454 gs flx
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
illumina solexa genetic analyzer
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
solid system
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
case study de novo genome sequencing
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)

case study de novo genome sequencing2
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

case study resequencing of genomes
Case study: resequencing of genomes

Bacterial genomes sequenced with

- Genome Analyzer (Illumina / Solexa)

- coverage between 33-fold and 70-fold

case study resequencing of genomes1
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

case study resequencing of genomes3
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

applications gs flx
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
applications genetic analyzer
Applications Genetic Analyzer
  • resequencing
  • ChIP
  • small RNAs
  • SAGE, SuperSAGE
  • CAGE
  • reference sequence is needed!
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