Next generation sequencing comparison of the technologies for genome sequencing
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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


Next generation sequencing comparison of the technologies for genome sequencing
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