Genome databases and webtools for genome analysis

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Genome databases and webtools for genome analysis

• Become familiar with microbial genome databases
• Use some of the tools useful for analyzing genome
• Visit sites used in lab exercise 2

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Major components of NCBI

• GenBank
• PubMed
• Entrez
• BLAST
• Conserved Domain Database (CDD)
• Cluster of orthologous groups (COGS)
• OMIM

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GenBank

• Database of DNA and protein sequences
• Searchable
• Caution Sequences deposited by the community,
  not curated for accuracy.
• RefSeq - verified by NCBI.

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Example of a GenBank record
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BLAST

• Basic Local Alignment Search Tool
• Comparing nucleotide sequences and protein
  sequences
• Microbial specific BLAST page
• Focus of a future lab

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OMIM

• Online Mendelian Inheritance in Man.
• Database that links diseases and genes

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TIGR

• Comprehensive microbial resource (CMR).
• Many genomes.
• Tools to analyze genomes.

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SubtiList

• Website for B. subtilis genome.
• Features
• Annotated genes
• Gene region display
• Updated similarity searches for every protein
• BLAST and pattern search capabilities
• Links to journal articles and protein databases

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RDP

• Ribosomal database project
• Curated at MSU
• Contains a compilation of all ribosomal DNA
  sequences (currently over 100,000).
• Second database contains information regarding
  copy number of ribosomal RNA.

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KEGG

• Kyoto Encyclopedia of Genes and Genomes
• Often changing database of gene content,
  metabolic pathways, etc.
• Excellent resource for reconstructing pathways in
  organism of interest.

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Genome sequencing and annotation
Week 2 reading assignments - pages 65-79,
110-122. Boxes 2.1, 2.2 and 2.3. Dont worry
about the details of HMM. Hughes Functional
Genomics Review.
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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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(No Transcript)
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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 segments 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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Finding genes in a genome sequence

• What to look for?
• Glimmer - HMM algorithm for identifying genes.
  (TIGR).
• ORF finder - NCBI.
• Most automated annotation engines have ORF
  finding capabilities.
• Much more difficult in eukaryotic genomes.