Functional Annotation
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Transcript Functional Annotation
Introduction to the GO:
a user’s guide
Iowa State Workshop
11 June 2009
All workshop materials are
available at AgBase.
Genomic Annotation
1.
2.
Genome annotation is the process of
attaching biological information to genomic
sequences. It consists of two main steps:
identifying functional elements in the
genome: “structural annotation”
attaching biological information to these
elements: “functional annotation”
biologists often use the term “annotation”
when they are referring only to structural
annotation
Structural
annotation:
DNA
annotation
CHICK_OLF6
Protein
annotation
TRAF 1, 2 and 3
Data from Ensembl Genome browser
TRAF 1 and 2
Functional annotation:
catenin
Structural & Functional Annotation
Structural Annotation:
Open reading frames (ORFs) predicted during genome
assembly
predicted ORFs require experimental confirmation
the Sequence Ontology (SO) provides a structured
controlled vocabulary for sequence annotation
Functional Annotation:
annotation of gene products = Gene Ontology (GO)
annotation
initially, predicted ORFs have no functional literature and
GO annotation relies on computational methods (rapid)
functional literature exists for many genes/proteins prior to
genome sequencing
GO annotation does not rely on a completed genome
sequence!
1.
2.
3.
4.
Provides structural annotation for
agriculturally important genomes
Provides functional annotation (GO)
Provides tools for functional modeling
Provides bioinformatics & modeling
support for research community
Introduction to GO
1.
2.
3.
pre-GO: managing large datasets
Bio-ontologies
the Gene Ontology (GO)
a GO annotation example
GO evidence codes
literature biocuration & computation analysis
ND vs no GO
sources of GO
1. pre-GO: managing
large datasets
AgBase User Support
Functional modeling training
Database ID mapping
approx.
Providing GO annotation for datasets/arrays
Assistance with GO modeling tools
Intermediary with between research community
and public databases
NCBI,
75% of requests
UniProtKB, GO Consortium
Computational assistance
Converting database accessions
UniProt database
Ensembl BioMart
Online analysis tools
DAVID, g:profiler, etc
AgBase database
ArrayIDer tool
More information about these
tools is available from the online
workshop resources.
1. UniProt ID Mapping
2. Ensembl BioMart
NOTE: Ensembl is scheduled to add plant & microbe species in 2009.
3. Online analysis tools
g:profiler conversion tool
http://biit.cs.ut.ee/gprofiler/gconvert.cgi
This tool works for all
species found in
Ensembl.
3. Online analysis tools
Database for Annotation, Visualization and Integrated Discovery
(DAVID)
http://david.abcc.ncifcrf.gov/conversion.jsp
This tool works for a
wide range of species.
4. AgBase: ArrayIDer
Contact AgBase to
request additional
species.
2. Bio-ontologies
Bio-ontologies
Bio-ontologies are used to capture biological
information in a way that can be read by both
humans and computers.
necessary for high-throughput “omics” datasets
allows data sharing across databases
Objects in an ontology (eg. genes, cell types, tissue
types, stages of development) are well defined.
The ontology shows how the objects relate to each
other.
Bio-ontologies:
http://www.obofoundry.org/
relationships
between terms
Ontologies
digital identifier
(computers)
description
(humans)
3. The Gene Ontology
Functional Annotation
Gene Ontology (GO) is the de facto method
for functional annotation
Widely used for functional genomics (high
throughput)
Many tools available for gene expression
analysis using GO
The GO Consortium homepage:
http://www.geneontology.org
GO Mapping Example
NDUFAB1 (UniProt P52505)
Bovine NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1, 8kDa
Biological Process (BP or P)
GO:0006633 fatty acid biosynthetic process TAS
GO:0006120 mitochondrial electron transport, NADH to ubiquinone TAS
GO:0008610 lipid biosynthetic process IEA
NDUFAB1
Molecular Function (MF or F)
GO:0005504 fatty acid binding IDA
GO:0008137 NADH dehydrogenase (ubiquinone) activity TAS
GO:0016491 oxidoreductase activity TAS
GO:0000036 acyl carrier activity IEA
Cellular Component (CC or C)
GO:0005759 mitochondrial matrix IDA
GO:0005747 mitochondrial respiratory chain complex I IDA
GO:0005739 mitochondrion IEA
GO Mapping Example
NDUFAB1 (UniProt P52505)
Bovine NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1, 8kDa
GO:ID (unique)
aspect or ontology
Biological Process (BP or P)
GO:0006633 fatty acid biosynthetic process TAS
GO:0006120 mitochondrial electron transport, NADH to ubiquinone TAS
GO:0008610 lipid biosynthetic process IEA
NDUFAB1
GO term name
GO:0005504
GO:0008137
GO:0016491
GO:0000036
Molecular Function (MF or F)
fatty acid binding IDA
NADH dehydrogenase (ubiquinone) activity TAS
oxidoreductase activity TAS
acyl carrier activity IEA
Cellular Component (CC or C)
GO:0005759 mitochondrial
matrix IDA code
GO evidence
GO:0005747 mitochondrial respiratory chain complex I IDA
GO:0005739 mitochondrion IEA
GO EVIDENCE CODES
Direct Evidence Codes
GO
Mapping
IDA
- inferred
fromExample
direct assay
IEP
- inferred(UniProt
from expression
NDUFAB1
P52505)pattern
IGIBovine
- inferred
fromdehydrogenase
genetic interaction
NADH
(ubiquinone) 1, alpha/beta subcomplex, 1, 8kDa
IMP - inferred from mutant phenotype
IPI - inferred from physical interaction
Biological Process (BP or P)
GO:0006633 fatty acid biosynthetic process TAS
Indirect Evidence Codes
GO:0006120 mitochondrial electron transport, NADH to ubiquinone TAS
inferred from literature
GO:0008610 lipid biosynthetic process IEA
IGC - inferred from genomic context
TAS - traceable author statement
Molecular Function (MF or F)
NAS - non-traceable author statement
GO:0005504 fatty acid binding IDA
IC - inferred by curator
GO:0008137 NADH dehydrogenase (ubiquinone) activity TAS
inferred by sequence analysis
GO:0016491 oxidoreductase activity TAS
NDUFAB1
RCA - inferred from reviewed GO:0000036
computational
acylanalysis
carrier activity IEA
IS* - inferred from sequence*
IEA - inferred from electronic annotation
Cellular Component (CC or C)
GO:0005759 mitochondrial matrix IDA
Other
ISS - inferred
from sequence
structural
similarity
GO:0005747
mitochondrial
respiratoryorchain
complex
I IDA
NR - not recorded (historical) GO:0005739 ISA
- inferred from
mitochondrion
IEA sequence alignment
ND - no biological data available
ISO - inferred from sequence orthology
ISM - inferred from sequence model
GO EVIDENCE CODES
Direct Evidence Codes
GO
Mapping
IDA
- inferred
fromExample
direct assay
IEP - inferred from expression pattern
IGI - inferred from genetic interaction
IMP - inferred from mutant phenotype
IPI - inferred from physical interaction
Biocuration of literature
• detailed function
• “depth”
• slower (manual)
Indirect Evidence Codes
inferred from literature
IGC - inferred from genomic context
TAS - traceable author statement
NAS - non-traceable author statement
IC - inferred by curator
inferred by sequence analysis
RCANDUFAB1
- inferred from reviewed computational analysis
IS* - inferred from sequence*
IEA - inferred from electronic annotation
Other
NR - not recorded (historical)
ND - no biological data available
ISS - inferred from sequence or structural similarity
ISA - inferred from sequence alignment
ISO - inferred from sequence orthology
ISM - inferred from sequence model
P05147
Biocuration of Literature:
detailed gene function
Find a paper
about the protein.
PMID: 2976880
Read paper to get experimental evidence of
function
Use most specific term
possible
experiment assayed kinase activity:
use IDA evidence code
GO EVIDENCE CODES
Direct Evidence Codes
GO
Mapping
IDA
- inferred
fromExample
direct assay
IEP - inferred from expression pattern
IGI - inferred from genetic interaction
IMP - inferred from mutant phenotype
IPI - inferred from physical interaction
Biocuration of literature
• detailed function
• “depth”
• slower (manual)
Indirect Evidence Codes
inferred from literature
IGC - inferred from genomic context
TAS - traceable author statement
NAS - non-traceable author statement
IC - inferred by curator
inferred by sequence analysis
RCANDUFAB1
- inferred from reviewed computational analysis
IS* - inferred from sequence*
IEA - inferred from electronic annotation
Other
NR - not recorded (historical)
ND - no biological data available
Sequence analysis
• rapid (computational)
• “breadth” of coverage
•less detailed
ISS - inferred from sequence or structural similarity
ISA - inferred from sequence alignment
ISO - inferred from sequence orthology
ISM - inferred from sequence model
Computational GO annotation (“breadth”)
ISO PIPELINE
accessions from your species
(species 1)
IEA PIPELINE
fasta file of sequences
(aa or nt)
public orthology
prediction tool(s)
InterPro analysis
(domains/motifs)
1:1 orthologs
domains/motifs in
sequence
existing GO
annotations
transfer GO annotation to
your species (ISO)
ga file
accessions
with no ISO
GO2InterPro
mapping file
assign GO (IEA)
no GO: “ND”
ga file
(integrate output into one ga file)
Ranjit Kumar
Unknown Function vs No GO
ND – no data
Biocurators
have tried to add GO but there is
no functional data available
Previously: “process_unknown”,
“function_unknown”, “component_unknown”
Now: “biological process”, “molecular
function”, “cellular component”
No annotations (including no “ND”):
biocurators have not annotated
1.
Primary sources of GO: from the GO
Consortium (GOC) & GOC members
2.
most up to date
most comprehensive
Secondary sources: other resources that use
GO provided by GOC members
public databases (eg. NCBI, UniProtKB)
genome browsers (eg. Ensembl)
array vendors (eg. Affymetrix)
GO expression analysis tools
Different tools and databases display the
GO annotations differently.
Since GO terms are continually changing
and GO annotations are continually
added, need to know when GO
annotations were last updated.
Secondary Sources of GO annotation
EXAMPLES:
public databases (eg. NCBI, UniProtKB)
genome browsers (eg. Ensembl)
array vendors (eg. Affymetrix)
CONSIDERATIONS:
What is the original source?
When was it last updated?
Are evidence codes displayed?
For more information about GO
GO Evidence Codes:
http://www.geneontology.org/GO.evidence.shtml
gene association file information:
http://www.geneontology.org/GO.format.annotation.shtml
tools that use the GO:
http://www.geneontology.org/GO.tools.shtml
GO Consortium wiki:
http://wiki.geneontology.org/index.php/Main_Page
All websites are available from the
workshop website & handout.