genome sequencing haixu tang school of informatics
TRANSCRIPT
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Genome sequencing
Haixu Tang
School of Informatics
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Methodology for DNA Sequencing
• The chain termination method (Sanger et al., 1977)
• The chemical degradation method (Maxam and Gilbert, 1977)
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Chain termination method
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Polyacrylamide gel electrophoresis
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Sequencing vectors
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PCR
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Sequencing primers
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Thermal cycle sequencing
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Automatic DNA
sequencing
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Pyrosequencing
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Sequencing by hybridization
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Whole genome shotgun
sequencing
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DNA cloning
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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.
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Assembly of the complete
Haemophilus influenzae
genome
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Problems with the shotgun approach
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Chromosome walking
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Chromosome walking by PCR
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Avoiding errors in WGS
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Scaffolds
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Human genome project
• 300,000 BACs “sequence-ready” map
• Shotgun sequencing of each BAC
• Order BACs to get the genome sequences