Genome sequencing Haixu Tang School of Informatics Methodology

Genome sequencing Haixu Tang School of Informatics

Methodology for DNA Sequencing • The chain termination method (Sanger et al. , 1977) • The chemical degradation method (Maxam and Gilbert, 1977)

Chain termination method

Polyacrylamide gel electrophoresis

Sequencing vectors

PCR

Sequencing primers

Thermal cycle sequencing

Automatic DNA sequencing

Pyrosequencing

Sequencing by hybridization

Whole genome shotgun sequencing

DNA cloning

Clone vectors • Bacteriophage P 1 vectors (Sternberg, 1990) can clone larger fragments of DNA, up to 125 kb using current technology. • Bacterial artificial chromosomes or BACs (Shizuya et al. , 1992) can be used to clone fragments of 300 kb and longer. • P 1 -derived artificial chromosomes or PACs (Ioannou et al. , 1994) combine features of P 1 vectors and BACs and have a capacity of up to 300 kb. • Fosmids (Kim et al. , 1992) contain the F plasmid origin of replication and a l cos site. They are less prone to instability problems.

Assembly of the complete Haemophilus influenzae genome

Problems with the shotgun approach

Chromosome walking

Chromosome walking by PCR

Avoiding errors in WGS

Scaffolds

Human genome project • 300, 000 BACs “sequence-ready” map • Shotgun sequencing of each BAC • Order BACs to get the genome sequences