Genome sequencing

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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