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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


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!



Thank you www gatc biotech com

Thank youwww.gatc-biotech.com


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