Genome sequencing and annotation

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Genome sequencing and annotation

• Week 2 reading assignment - pages 63-78, 93-98,
  105-111. Boxes 2.1 and 2.3 - dont worry about
  details of similarity scoring and hidden markov
  models.

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• Sequencing - dideoxy method for DNA sequencing.
• Methods for sequencing genomes.
• Methods for finding and annotating genes in
  microbial genomes.

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Dideoxy sequencing (Sanger method)

• Developed by Frederick Sanger (for which he won
  his second Nobel Prize in 1980).

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Two types of labeling

• Radioactive
• 32P, 35S
• Run out each dideoxy base in a separate reaction,
  lane on a gel.
• No longer used
• Fluorescent
• Four different fluorophores for each base
• Can be mixed.
• Chromatograms - GTSF

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Cycle sequencing
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Phred

• Method for automated quality assessment of DNA
  sequence traces.
• Variance in peak spacing in 7 peak window
• Ratio of largest uncalled peak to smallest called
  peak in 7 and 3 peak windows.
• Number of bases between current base and nearest
  unresolved base.
• Phred score 10 x (-log(P)).
• Phred scores of 20 or higher are considered good
  calls. Why?

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Sequencing of genomes

• Hierarchical or contig based sequencing
• Clone smaller seqments of the genome.
• Labor intensive, slow
• Not needed for sequencing microbial genomes
• Shotgun method
• Randomly clone and sequence 1.5-2 kb fragments of
  DNA. 5-10 fold coverage.
• Computationally intensive.

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Sequence assembly

• Focus of this weeks lab exercise
• Algorithms to align and edit multiple sequences
• Phrap and Consed
• Sequencher (commercial) for lab.
• List of features of good sequence editors on page
  70.

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Sequenced microbial genomes

• Haemophilus influenzae - 1995.
• Over 250 microbes currently sequenced.
• Why sequence so many microbial genomes?
• Develop technology for human genome project
• Examine the genomes of a wide range of microbes
• Novel drug/vaccine targets
• Identify new agricultural and industrial
  important enzymes
• Comparative genomics/evolution
• Sequence microbes that have no genetics
• Sequence microbes we cant culture - metagenomics
• Because we can.
• Financially feasible to sequence entire genomes.

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Finding functional features in a microbial genome.

• Genes
• Origin of replication
• rRNA operons, tRNAs
• Promoters
• Transcription terminators
• Horizontially transferred DNA
• GC content