FlyBase:Gene Ontology (GO) Annotation

G.3. Classification of Gene Products using Gene Ontology (GO) terms

FlyBase uses Gene Ontology (GO) controlled vocabulary (CV) terms for cellular component, biological process and molecular function to describe properties of gene products. Although GO terms are intended to describe the properties of gene products, FlyBase currently assigns GO terms to genes rather than protein or RNA.

FlyBase is one of the founding members of the Gene Ontology (GO) Consortium and follows the general guidelines for GO annotation as described in the GO documentation.

Also, see the related video tutorial Finding related genes in FlyBase: The Gene Ontology.

G.3.1. FlyBase GO data

GO data is displayed in the Gene Ontology: Function, Process, and Cellular component section of individual Gene Reports.

In addition, the current release of GO data for all Drosophila melanogaster FlyBase genes can be found in the tab delimited text file gene_association.fb.

The following provides a brief description of the columns in the gene_association.fb file.

Gene Association file columns

1. DB The database contributing the gene_association file

FB File: always "FB" for gene_association.fb.

2. DB_Object_ID A unique identifier in the database for the item being annotated.

FB File: This is always the primary FlyBase identifier number for a Drosophila gene.

Example: FBgn0000490

3. DB_Object_Symbol

A (unique and valid) symbol to which the DB_Object_ID is matched.

FB File: This is always the valid gene symbol for a Drosophila gene.

Example: dpp

4. Qualifier (this field is optional)

One or more of 'NOT', 'contributes_to' or 'colocalizes_with' as qualifier(s) for a GO annotation.

Multiple qualifiers are separated by a pipe (|).

FB File: 'contributes_to' or 'colocalizes_with' are not currently

displayed in gene_association.fb, but they will be displayed in the

next release of the FlyBase gene_association file.

5. GO ID

The unique GO identifier for the GO term attributed to the DB_Object_ID.

Example: GO:0005160

6. DB:Reference

The unique identifier for the reference to which the GO annotation is attributed.

FB File: Each FlyBase reference including published literature,

conference abstracts, personal communications, sequence records and

computer files has a unique 7 digit identifier (an FBrf). Where this

reference is a published paper with a PubMed identifier, the PubMed ID

is also listed in column 6, separated from the FBrf with a pipe (|).

Example: FB:FBrf0136863|PMID:11432817

7. Evidence

The evidence code for the GO annotation; one of IMP, IGI, IPI, ISS, IDA, IEP, IEA, TAS, NAS, ND, IC, RCA

8. With (or) From

FB File: This column contains the identifier for annotations where the

evidence code is IGI, IPI, ISS, IEA or IC. For IGI the database gene

symbol and identifier is listed. For ISS and IPI the identifier can be a gene

symbol and identifier, or a sequence (protein or nucleic acid)

identifier. For IC, the GO identifier of the term used as the basis of

a curator inference is given. With statements for IC are not currently

displayed in gene_association.fb, but they will be displayed in the

next release of the FlyBase gene_association file.

IGI example: FLYBASE:rpr; FB:FBgn0011706

ISS example: UniProt:P35569

ISS example: EMBL:AF064523

ISS example: SGD_LOCUS:COP1; SGD:S0002304

IC example: GO:0045298

9. Aspect

Which ontology the GO term belongs to: Function (F), Process (P) or Component (C).

Example: P

10. DB_Object_Name

FB File: The full name of the FlyBase gene.

Example: decapentaplegic

Where a FlyBase gene has no full name (eg Pten), this field is left blank.

11. DB_Object_Synonym

Alternative names by which the database object is known.

FB File: Multiple synonyms of a FlyBase gene are separated by a pipe (|).

Example: M(2)LS1|shortvein|Dm-DPP|dpp|Dpp|DPP|CG9885|

TGF-beta|TGF-&bgr;|TGF-b|Hin-d|l(2)10638|shv|

DPP-C|ho|M(2)23AB|blk|l(2)22Fa|l(2)k17036|Tg|TGF&bgr;

12. DB_Object_Type

The type of object being annotated. Always a gene for FlyBase data.

FB file: always "gene" for gene_association.fb.

13. taxon

The taxonomic identifier of the species encoding the gene product

Example: taxon:7227

14. Date

The date of last annotation update, in the format 'YYYYMMDD'. At

present this date is the same for all annotations and corresponds to

the date of the latest FlyBase update; we are in the process of

changing our system so that dates more accurately reflect the date the

annotation is made.

Example: 20040821

15. Assigned_by

The source of the GO annotation.

FB File: One of either FB or UniProtKB.

The latest version of this data is also available for download here from the Gene Ontology consortium site. The accompanying README document includes a detailed description of the file format, FlyBase GO annotation policy and sources used for FlyBase GO annotations.

Note that the GO data available from FlyBase will not necessarily be identical to that found on the GO website. GO validate the data FlyBase submits and remove lines of data that are no longer valid e.g. when a GO term becomes obsolete.

QueryBuilder can be used to identify all the genes associated with a particular GO term. The AmiGO and QuickGO browsing tools can be used to find GO terms of interest. See video tutorial Finding related genes in FlyBase: The Gene Ontology.

G.3.2. Evidence

Evidence for a GO term consists of an evidence code that describes the type of analysis carried out together with, in some cases, a reference to another database object in that supports the evidence (see with/from Supporting Evidence below).

Evidence codes The Gene Ontology Guide to GO Evidence Codes contains comprehensive descriptions of the evidence codes used in GO annotation. FlyBase uses the following evidence codes when assigning GO data:

inferred from experiment (EXP)

inferred from mutant phenotype (IMP)

inferred from genetic interaction (IGI)

inferred from direct assay (IDA)

inferred from physical interaction (IPI)

inferred from expression pattern (IEP)

inferred from high throughput experiment (HTP)

inferred from high throughput mutant phenotype (HMP)

inferred from high throughput genetic interaction (HGI)

inferred from high throughput direct assay (HDA)

inferred from expression pattern (HEP)

inferred from sequence or structural similarity (ISS)

inferred from sequence orthology (ISO)

inferred from sequence alignment (ISA)

inferred from biological aspect of ancestor (IBA)

inferred from electronic annotation (IEA)

inferred from reviewed computational analysis (RCA)

traceable author statement (TAS)

non-traceable author statement (NAS)

inferred by curator (IC)

no biological data available (ND)

G.3.2.1. Use of evidence codes

Consistent with the aims of the GO reference genome project, FlyBase prefers to assign GO terms based on experimental evidence codes (IMP, IGI, IDA, IPI, IEP). Infrequently, GO annotations will be associated with genes frim high throughput (HTP) experiments. The HTP experiments are only annotated when the satisfy a number of rules, to ensure that there are minimal false positives propagated by GO annotation. These experimental annotations are marked by the evidence codes HTP, HDA, HMP, HGI and HEP, so that they can be distinguished from low-throughput and generally hypothesis-driven experiments.

Evidence codes based on computer predictions (IEA, RCA), author statements (NAS, TAS) and curator inference (IC) will continue to be used in the absence of experimental data for the same or a more specific GO term. However, we aim to remove GO data with these codes when experimental evidence for the term is curated.

The evidence code ND (no biological data available) is used in combination with the root GO terms "molecular_function ; GO:0003674", "biological_process ; GO:0008150" or "cellular_component ; GO:0008372". In FlyBase the use of any of these three GO terms, attributed to reference FBrf0159398 and supported by the ND evidence code, signifies that a curator has examined the available literature and sequence for this gene and that, as of the date of the annotation to the term, there is no information supporting an annotation to a specific GO term.

Additional information about the way FlyBases uses evidence codes can be found in the README document.

with/from Supporting Evidence Some evidence codes (IGI, IPI, ISS, IEA, IC) are used in conjunction 'with' supporting data in the form of a reference to another database object. These objects are identified by their database abbreviation followed by a colon and the unique identifier for the object in that database. A list of current database abbreviations can be found in the GO.xrf_abbs file. See the GO Annotation Guide for more details.

ISS and IEA 'with'

FlyBase captures GO data based on similarity to other gene products that are known to have that attribute. Since October 1st 2006, it has been mandatory for ISS annotations to include an identifier for the sequence used to make the annotation; earlier FlyBase ISS annotations that do not include identifiers will be updated gradually. In line with current guidelines for reference genomes, curators now check that the similar sequence can be annotated to the GO term with experimental evidence (IDA, IMP, IGI, IPI, IEP) before making an ISS annotation. This policy was adopted to avoid circular similarity-based annotations. Consequently, GO terms are not curated based multiple sequence alignments if none of the sequences in the alignment have been experimentally verified. Annotations made before October 2006 have not necessarily been checked in this way.

For example, the Drosophila gene bigmax is annotated with the GO term 'regulation of transcription' based on sequence similarity to Max. This annotation is legitimate because Max has been shown to regulate transcription in a direct assay.

The combined evidence appears on the gene report in the format:

inferred from sequence or structural similarity with FLYBASE:Max; FB:FBgn0017578

In this case we have give two identifiers (symbol and gene ID) for the same sequence; identifiers for the same sequence are separated by a semi-colon. If more than one sequence is used to make the annotation then the identifiers for the different sequences are separated by a comma. Note that this use of multiple identifiers is a different to that for IGI and IPI.

Where the database object used to to make IEA annotations can be identified then this is included in the same way. However, the majority of FlyBase annotations with IEA do not yet include such a reference. Most IEA annotations in FlyBase are based on the presence of protein domains that are mapped to GO terms. The identifiers for the protein domains will be included in future releases.

IGI and IPI 'with'

For both IGI and IPI there is a special meaning and All annotations inferred from genetic interaction (IGI) include an identifier for the interacting gene. If the GO term is inferred based on multiple genes interacting simultaneously then all interacting genes are identified using 'with' (separated by commas). However, if the GO term is inferred from multiple pairwise interactions these are treated as separate pieces of experimental evidence and appear with separate evidence codes on the gene report.

For example, Bruce is annotated with the GO term 'programmed cell death' based on two different pairwise genetic interaction experiments; the evidence appears on the gene report as:

inferred from genetic interaction with FLYBASE:grim; FB:FBgn0015946 AND inferred from genetic interaction with FLYBASE:rpr; FB:FBgn0011706

Contrast this with, the following which would imply that all three genes had to interact together to provide evidence for the annotation:

inferred from genetic interaction with FLYBASE:grim; FB:FBgn0015946, FLYBASE:rpr; FB:FBgn0011706

Similar notation is used for IPI where the interacting gene product is identified using 'with'. Where several gene products interact simultaneously they are recorded in a single annotation (separated by commas after the evidence code). Pairwise physical interactions are recorded independently with using separate evidence codes.

IC 'from'

Evidence inferred by curator (IC) is the case that includes 'from'. Curators use this code for those cases where an annotation is not supported by any evidence, but can be reasonably inferred by from other GO annotations, for which evidence is available. The object identified in the IC evidence is always a GO term identifier.

For example, a protein shown to have transcription factor activity in a direct assay could be annotated with the GO term 'general RNA polymerase II transcription factor' (GO:0016251). In the absence of any evidence for the cellular location of that protein, if would be reasonable for the the curator to infer that it is (at least sometimes) located in the nucleus. This would lead the the annotation, nucleus inferred by curator from GO:0016251; the annotation is attributed to the reference that contains evidence for transcription factor activity.

G.3.2.2. Use of Qualifiers

Qualifiers are used as flags that modify the interpretation of an annotation. Allowable values are NOT, contributes_to, and colocalizes_with. On the gene report page, qualifiers precede the GO term in the CV column. More information about using qualifiers is available in the GO Annotation Guide.

NOT

NOT may be used with terms from any of the three GO ontologies (cellular component, biological process, molecular function).

NOT is used to make an explicit note that the gene product is not associated with the GO term. This is particularly important in cases where associating a GO term with a gene product should be avoided (but might otherwise be made, especially by an automated method).

For example, if a protein has sequence similarity to an enzyme such as galactosyltransferase, but has been shown experimentally not to have the galactosyltransferase activity, it can be annotated as NOT galactosyltransferase activity (GO molecular function term: GO:0008378).

NOT can also be used when a cited reference explicitly says (e.g. "our favorite protein is not found in the nucleus"). Prefixing a GO term with the string NOT allows curators to state that a particular gene product is NOT associated with a particular GO term. This usage of NOT was introduced to allow curators to document conflicting claims in the literature.

Note that NOT is used when a GO term might otherwise be expected to apply to a gene product, but an experiment, sequence analysis, etc. proves otherwise; it is not generally used for negative or inconclusive experimental results.

colocalizes_with

colocalizes_with is used only with cellular component terms.

Gene products that are transiently or peripherally associated with an organelle or complex are annotated to the relevant cellular component term, using the colocalizes_with qualifier. This qualifier is also be used in cases where the resolution of an assay is not accurate enough to say that the gene product is a bona fide component member.

contributes_to

contributes_to is used only with molecular function terms.

An individual gene product that is part of a complex is annotated to terms that describe the function of the complex. Many such function annotations include the qualifier contributes_to:

Annotating individual gene products according to attributes of a complex is especially useful for molecular function annotations in cases where a complex has an activity, but not all of the individual subunits do. (For example, there may be a known catalytic subunit and one or more additional subunits, or the activity may only be present when the complex is assembled.) Molecular function annotations of complex subunits that are not known to possess the activity of the complex include the qualifier contributes_to.

Note that contributes_to is not used to annotate a catalytic subunit. Furthermore, contributes_to may be used for any non-catalytic subunit, whether the subunit is essential for the activity of the complex or not.