genome sequencing haixu tang school of informatics
TRANSCRIPT
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 P1 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.
• P1-derived artificial chromosomes or PACs (Ioannou et al., 1994) combine features of P1 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