Genome Annotation Continued

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Genome Annotation Continued

• This weeks lab.
• Genome annotation - web based databases for
  assigning gene function.

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Last weeks lab

• E-value
• Score
• Blastx
• Taxonomy

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Lab

• Sequence assembly and analysis
• Assemble individual sequence reads
• Phred 30 - good or bad?

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Linking Protein Sequence, Structure, and Function
Protein sequences
Protein
CDD Conserved functional domains in proteins
represented by a PSSM
Domains
PSI-BLAST, RPS-BLAST, CDART
3D Domains
NCBI Field Guide
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Position Specific Substitution Rates
Active site serine
Weakly conserved serine
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Position Specific Score Matrix (PSSM)
A R N D C Q E G H I L K M
F P S T W Y V 206 D 0 -2 0 2 -4 2 4
-4 -3 -5 -4 0 -2 -6 1 0 -1 -6 -4 -1 207 G
-2 -1 0 -2 -4 -3 -3 6 -4 -5 -5 0 -2 -3 -2 -2
-1 0 -6 -5 208 V -1 1 -3 -3 -5 -1 -2 6 -1
-4 -5 1 -5 -6 -4 0 -2 -6 -4 -2 209 I -3 3
-3 -4 -6 0 -1 -4 -1 2 -4 6 -2 -5 -5 -3 0 -1
-4 0 210 S -2 -5 0 8 -5 -3 -2 -1 -4 -7 -6
-4 -6 -7 -5 1 -3 -7 -5 -6 211 S 4 -4 -4 -4
-4 -1 -4 -2 -3 -3 -5 -4 -4 -5 -1 4 3 -6 -5 -3
212 C -4 -7 -6 -7 12 -7 -7 -5 -6 -5 -5 -7 -5 0
-7 -4 -4 -5 0 -4 213 N -2 0 2 -1 -6 7 0
-2 0 -6 -4 2 0 -2 -5 -1 -3 -3 -4 -3 214 G
-2 -3 -3 -4 -4 -4 -5 7 -4 -7 -7 -5 -4 -4 -6 -3
-5 -6 -6 -6 215 D -5 -5 -2 9 -7 -4 -1 -5 -5
-7 -7 -4 -7 -7 -5 -4 -4 -8 -7 -7 216 S -2 -4
-2 -4 -4 -3 -3 -3 -4 -6 -6 -3 -5 -6 -4 7 -2 -6
-5 -5 217 G -3 -6 -4 -5 -6 -5 -6 8 -6 -8 -7
-5 -6 -7 -6 -4 -5 -6 -7 -7 218 G -3 -6 -4 -5
-6 -5 -6 8 -6 -7 -7 -5 -6 -7 -6 -2 -4 -6 -7 -7
219 P -2 -6 -6 -5 -6 -5 -5 -6 -6 -6 -7 -4 -6 -7
9 -4 -4 -7 -7 -6 220 L -4 -6 -7 -7 -5 -5 -6
-7 0 -1 6 -6 1 0 -6 -6 -5 -5 -4 0 221 N
-1 -6 0 -6 -4 -4 -6 -6 -1 3 0 -5 4 -3 -6 -2
-1 -6 -1 6 222 C 0 -4 -5 -5 10 -2 -5 -5 1
-1 -1 -5 0 -1 -4 -1 0 -5 0 0 223 Q 0 1
4 2 -5 2 0 0 0 -4 -2 1 0 0 0 -1 -1 -3 -3
-4 224 A -1 -1 1 3 -4 -1 1 4 -3 -4 -3 -1
-2 -2 -3 0 -2 -2 -2 -3
Serine is scored differently in these two
positions
Active site nucleophile
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Hidden Markov Models

• A statistical model that can be applied to any
  system that is represented as a discrete state.
• Applies to protein and nt sequences.
• Can be thought of much like PSSMs used in
  PSI-BLAST.
• After several interations.
• Are used in gene finding and protein profile
  analysis.

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Uses of HMMs in protein function analysis.

• TIGRFAMs
• Strive to annotate function of an entire protein
• PFAMs
• Strive to annotate domains of proteins.

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Homologs, orthologs, and paralogs.

• Homologous genes are genes that share a common
  evolutionary ancestor.
• Orthologs are genes found in different organisms
  that arose from a common ancestor. Speciation.
• Paralogs are genes found in the same organism
  that arose from a common ancestor. Duplication
  could have occurred in the species or earlier,
  often have diverged in function

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Orthologs may differ in function!
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TIGRFAM

• Curated such that proteins in a TIGRFAM should
  have the same function if they are equivalogs.
• Proteins have identity over their entire length.
• Equivalog family all proteins that are
  conserved with respect to function since their
  last common ancestor.
• Superfamily - all proteins with homology but may
  have different biological functions.
• Subfamily - incomplete set of proteins with
  homology - may have diverse biological functions.

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PFAM

• More likely to describe a protein domain rather
  than a family.
• Pfams will not overlap.
• Crosslisted in TIGRFAM page.
• 70 of proteins in SWISS-Prot have a Pfam match.

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COGs

• Cluster of orthologous groups
• Pairwise comparison of orthologs from many
  bacterial genomes.
• Suggests function only (book example).

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Gene Ontology (GO)

• The goal of the Gene Ontology project is to
  produce a controlled vocabulary that can be
  applied to all organisms even as knowledge of
  gene and protein roles in cells is accumulating
  and changing.
• Biological process, Molecular function, Cellular
  component

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Literature Curation

• Saccharomyces genome database (SGD) for example.
• Manual curation of the literature for
  experimental evidence linking function to
  annotation.

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Additional databases

• SMART - Simple Modular Architecture Research
  Tool.
• PROSITE - Protein motifs
• PRODOM - A database based on PSI-BLAST PSSMs.
• InterPro - A database that brings together many
  of the above databases so that you can search
  them all at once.
• Others.

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CDD

• Conserved domain database - linking all of this
  information together.
• Consists of SMART, Pfam, and COGs (KOGs).
  Searchable directly - automatically searched by
  BLAST.
• Linked to CDART - allows the identification of
  proteins with a similar domain architecture.

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Bottom line about databases

• Are useful tools in assigning possible functions.
• Be careful about annotations
• example -proteins in the same COG can be
  orthologs that have evolved different functions.
• Many annotations are not backed up by
  experimental data.
• Some databases are automated - have not been
  checked for accuracy.

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Annotation can not be guaranteed without
experimental evidence.

• Functional genomics