BME 130 – Genomes

Lecture 4

Sequencing technology IINext generation

sequencing

Figure 4.2 Genomes 3 (© Garland Science 2007)

Figure 4.3a Genomes 3 (© Garland Science 2007)

Roche / 454

454 Process Overview

1) Prepare Adapter Ligated ssDNA Library

2) Clonal Amplification on 28 µ beads

4) Perform Sequencing by synthesison the 454 Instrument of ~250,000 molecules

3) Load beads and enzymes in PicoTiter Plate™

Key sequence = TCAG for identifying wells and calibration

TACG

Flow Order

454 base calling

Limitations of Roche/454

Solexa / Illumina

Limitations of Solexa/Illumina

ABI/SOLiD

Limitations of ABI/SOLiD

Applications ofhigh-throughput sequencing

Genome assembly

CHiP-Seq

Johnson et al., Science 8 June 2007:Vol. 316. no. 5830, pp. 1497 - 1502

RNASeq

Cloonan et al., Genome Biology 2008, 9:234