Transcript Lec-Functional Annotation and Functional Enrichment2010

Functional Annotation and
Functional Enrichment
Annotation
• Structural Annotation – defining the boundaries of features of
interest (coding regions, regulatory elements, functional
RNAs, etc).
– Ab initio – computationally predicted
– Comparative – based on similarity to other genes or genomes
– Experimental – transcript sequencing
• Functional Annotation – attaching meaning to the features
(names, product, activity, biological role, etc.)
– Sequence homology
– Structural similarity or structural features
– Experimental data – gene or protein expression patterns
Functional Annotation
Manual
• Slow
• Costly
• Inconsistent quality
• Inconsistent coverage
across genome
• Rich content
• Error correction
Automated
• Fast
• Cheap?
• Consistent quality
• Complete coverage across
genome
• Improving in content
• Updateable
Home many ways are there to say the
same thing?
• Quick survey of GenBank lacI product annotations in 48 bacteria:
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Lactose operon repressor (20)
DNA-binding transcriptional repressor (14)
transcriptional regulator LacI family (5)
lac operon repressor (2)
transcriptional repressor of the lac operon (2)
lac repressor (1)
LacI (1)
putative transcriptional regulator (1)
transcriptional repressor of lactose catabolism (1)
transcriptional repressor of lactose catabolism (GalR/LacI family) (1)
* Excluding differences in capitalization
The Gene Ontology (GO)
• Goal = consistent annotation of gene products within
and between organisms
• Gene Ontology Consortium began as a collaboration
among model organism dbs (FlyBase, SGD, MGD).
Now includes larger number of members and
interest groups
• Ontology = A formal representation of concepts and
the relationships among them
Gene Ontology
The 3 GO Ontologies
• Molecular Function (8,360 terms)
• Biological Process (14,898 terms)
• Cellular Component (2,110 terms)
• GO Term = an entry in an ontology, composed of a
unique identifier (GO:000001), definition and
“synoynms”
CC
• A cellular component is just that, a
component of a cell, but with the proviso that
it is part of some larger object;
• this may be an anatomical structure (e.g.
rough endoplasmic reticulum or nucleus) or a
gene product group (e.g. ribosome,
proteasome or a protein dimer).
BP
• A biological process is series of events accomplished by
one or more ordered assemblies of molecular functions.
• Examples of broad biological process terms are cellular
physiological process or signal transduction. Examples of
more specific terms are pyrimidine metabolic process or
alpha-glucoside transport.
• It can be difficult to distinguish between a biological
process and a molecular function, but the general rule is
that a process must have more than one distinct steps.
• A biological process is not equivalent to a pathway; at
present, GO does not try to represent the dynamics or
dependencies that would be required to fully describe a
pathway.
MF
• Molecular function describes activities, such as catalytic or
binding activities, that occur at the molecular level. GO
molecular function terms represent activities rather than the
entities (molecules or complexes) that perform the actions, and
do not specify where or when, or in what context, the action
takes place. Molecular functions generally correspond to
activities that can be performed by individual gene products,
but some activities are performed by assembled complexes of
gene products.
• Examples of broad functional terms are catalytic activity,
transporter activity, or binding; examples of narrower
functional terms are adenylate cyclase activity or Toll receptor
binding.
• It is easy to confuse a gene product name with its molecular
function, and for that reason many GO molecular functions are
appended with the word "activity".
Annotation File Format
Evidence Codes
• Experimental Evidence Codes
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EXP: Inferred from Experiment
IDA: Inferred from Direct Assay
IPI: Inferred from Physical Interaction
IMP: Inferred from Mutant Phenotype
IGI: Inferred from Genetic Interaction
IEP: Inferred from Expression Pattern
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ISS: Inferred from Sequence or Structural Similarity
ISO: Inferred from Sequence Orthology
ISA: Inferred from Sequence Alignment
ISM: Inferred from Sequence Model
IGC: Inferred from Genomic Context
RCA: inferred from Reviewed Computational Analysis
• Computational Analysis Evidence Codes
Evidence Codes
• Author Statement Evidence Codes
– TAS: Traceable Author Statement
– NAS: Non-traceable Author Statement
• Curator Statement Evidence Codes
– IC: Inferred by Curator
– ND: No biological Data available
• Automatically-assigned Evidence Codes
– IEA: Inferred from Electronic Annotation
• Obsolete Evidence Codes
– NR: Not Recorded
What is the source of automated
annotations?
• Integrated automated annotation systems
combine a variety of analysis types
• Comparison to databases protein and/or
domain families with defined functions (COGs,
NCBI CDD, PFAM, ProSite, etc.)
• Structural characteristic predictions
• Sequence characteristic predictions
InterPro: www interface
InterPro
InterPro release 16.0 contains
15045 entries:
Active sites
34
Binding sites
22
Domains
4676
Families
10060
PTMs
18
Repeats
235
Database
All Signatures
Integrated
PANTHER
30128
2061
Pfam
8957
8957
PIRSF
1748
1499
PRINTS
1900
1898
ProDom
3538
1041
PROSITE
1319
1319
SMART
724
721
TIGRFAMs
2949
2933
Gene3D
2147
783
SUPERFAMILY
1538
463
Sample InterPro Family
InterPro is one source of IEAs
On a genome scale
• Assign all genes to Interpro families
• Obtain GO terms (IEA evidence) linked to the
Interpro term
• Use these to find patterns in large gene lists
– Experimental ( genes upregulated in array exp)
– Comparative (genes with/without orthologs)
Enrichment
• Find categories (InterPro, GO) that are overrepresented in a subset of genes relative to
the background (genome?) as a whole
• Example: 40% of the genes that distinguish
between two strains of E. coli are mobile
elements. Is this more than I expect based on
random chance if 10% of the genome as a
whole is mobile elements.
Hypergeometric Distribution
• describes the number of successes in a sequence of n draws
from a finite population without replacement
• Black and white balls in an urn
• Genes with an ortholog and genes without an ortholog
• Genes differentially expressed, genes unchanged
Comparison of 68
enrichment analysis
tools available in 2008