next generation sequencing: comparison of the technologies for genome sequencing

© 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


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



Overview

Illumina / Solexa Genetic Analyzer2000 Mb / run

Applied Biosystems ABI 3730XL1 Mb / day

Roche / 454 Genome Sequencer FLX100 Mb / run

Applied BiosystemsSOLiD3000 Mb / run



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

• 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

• 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

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: hard / full stops



Case study: hard / full stops

454



Flowgrams in SeqMan



Homopolymers




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

Bacterial genomes sequenced with

- Genome Analyzer (Illumina / Solexa)

- coverage between 33-fold and 70-fold




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




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

• 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

• resequencing

• ChIP

• small RNAs

• SAGE, SuperSAGE

• CAGE

• reference sequence is needed!



Applications Genetic Analyzer


Thank you

www.gatc-biotech.com

next generation sequencing: comparison of the technologies for genome sequencing

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