Viewing & Getting GOCOST Functional Modeling Workshop22-24 April, Helsinki

Summary1. Ontology Browsers

• QuickGO, AmiGO – searching for GO Terms, getting GO• Plant Ontology Browser

2. Finding/ Adding GO for Functional Modeling.• GOProfiler – summary of GO for your species• GORetriever – gets evisting GO• GOanna – adds GO (Blast)• Adding GO for large datasets

3. Array annotation4. Using added GO in GO Enrichment Analysis.

GO Browsers• QuickGO Browser (EBI GOA Project)

• http://www.ebi.ac.uk/ego/• protein annotations• search by GO Term or by UniProt ID

• AmiGO Browser (GO Consortium Project)• http://amigo.geneontology.org/cgi-bin/amigo/

go.cgi• search by GO Term or by accession

http://www.ebi.ac.uk/QuickGO/

Example: QuickGO Browser

QuickGO Features• Searching for gene products

• Can use gene/protein gene names, but better to use accessions.• Works off UniProt accessions/IDs• Can enter multiple accessions (separated by a space).

• Can search for GO Terms• Has autocomplete• Provides ranked list of matches• Matches are also grouped by BP, CC, MF• GO Terms – definitions, annotations, parents, children terms.

• Advanced filtering options.• Download as protein lists or gene association file format.

record information

annotation datanumber of annotations

Add taxon ID for horse.

(Use to find taxon ID for your species.)

http://amigo.geneontology.org

Example: AmiGO Browser

AmiGO Features• Need to select either a gene product of GO Term search.• Searching for gene products

• Can use gene/protein gene names, but better to use accessions.• Works off multiple accessions/IDs• Only accepts a single accession, not a list.• View information about gene product & about annotations for

that gene product.• Can search for GO Terms

• Large numbers of GO annotations are truncated.• Some filtering options.

• Filter by ontology or evidence code.• Filter by database or species.

• Download as sequences or gene association file.

Plant Ontology (PO) Browser

• describes plant anatomy and morphology and stages of development for all plants

• Plant Anatomy• e.g., plant structures (PO:0009011) such as plant organ

(PO:0009008), plant cell (PO:0009002), whole plant (PO:0000003), portion of plant tissue (PO:0009007), and vascular system (PO:0000034), etc.

• Plant Structure Development Stage• e.g., plant tissue development stage (PO:0025423), leaf

development stage (PO:0001050), whole plant development stage (PO:0007033), seed development stage (PO:0001170), and sporophyte development stage (PO:0028002), etc.

http://www.plantontology.org/

Ontology Browsers

• Use to identify specific ontology terms of interest.

• Use to download specific annotation files• for specific gene lists• for species

use as input for GO or PO expression analysis

Tutorial 1.

• Familiarizing your self with ontology browsers.

OR• Use browsers to look for GO/PO for

accessions from your own data set.

2. Finding/ Adding GO for Functional Modeling

How much GO is

available for your species?

How much GO is

available for your data set?

How much of this is in the tool(s) you

want to use?

Do you need to add GO?

GOProfiler GORetriever Last update?Source?

GOanna, Blast2GO,

etc

GOProfilerGOProfiler allows you get an overview of what GO annotation

exists for the species you are interested in.

Number of proteins is based upon GO Consortium records for these species.

Species with only IEA annotations do not have an active GO annotation project GO provided

automatically by EBI GOA Project.

GORetriever Allows you to get existing GO annotations for a specific set of

gene products. Accepts a text file of accessions or IDs. Returns GO annotations, list of accessions that have no GO and

a GO Summary file.

Input file – text file of return separated accessions.

GORetriever Results

GORetriever Results

GORetriever Resultsadd GO to this list using GOanna or

Blast2GO

GORetriever Results do functional grouping using GOSlimViewer

• only returns existing GO • only accepts limited accession types

• GOanna does a Blast search against existing GO annotated products.

• allows you to quickly transfer GO to gene products where they have similar sequences

• accepts fasta files

Incorrect email address – you will not receive your results!

Contact AgBase if you have not received results after 24-48h.

GOanna ResultsIf you enter an incorrect email address – you will not receive your results!

Contact AgBase if you have not received results after 24-48h.

query IDs are hyperlinked to BLAST data(files must be in the same directory)

*WHAT IS A GOOD ALIGNMENT?

1. Manually inspect alignments and delete any lines where there is not a good alignment*.2. Add this additional annotation to the annotations from GORetriever.

GOanna2gaNew to AgBase: an online script to convert your GOanna file to a gene association file format.

• add manually checked GOanna annotations to a GORetriever file

Tutorial 2

• Getting GO.• GOProfiler – check what is available• GORetriever – get existing GO• GOanna – add GO annotations

Note - you will use Blast2GO to add additional GO annotations to your data sets tomorrow.

OR• Getting existing GO & adding additional GO to your

own data set.

Some limitations of GOanna:

• BLAST analysis is slow – results emailed• limit to 5,000 inputs or an overall file size of 6Mb• limit to 3 jobs submitted/user at one time

How do I do to get GO for my 50,000 RNA-Seq dataset?

• 50 x GOanna submissions + manual interpretation of results – impractical and slow!!

• ALTERNATIVELY: Contact AgBase• we use internal GO annotation pipelines/queuing• We can help customize databasess• GO can be kept private and released after

publication

How do I do to get GO for my 50,000 RNA-Seq dataset?

• GOanna is being deployed on the iPlant discovery environment• increased computing capacity• faster Blast searches• no limitations on file number or size

http://www.iplantcollaborative.org/

GO annotation of RNA-Seq data1. Retrieve any existing GO annotation for gene products

• Genome2Seq: Rapidly retrieves a fasta file of sequences and GO based on genome co-ordinates generated from RNA-Seq data.

2. InterProScan – identifies functional motifs and domains• Can be mapped to GO terms (IEA)• VERY computer intensive – do this on HPC resources; being

implemented on iPlant• Improved results if transcripts are translated (e.g. EMBOSS)

3. BLAST based similarity transfer (ISA)• e.g. Blast2GO, GOanna• Should only transfer GO annotations based upon direct experimental

evidence codes.• Need to test sample set to determine “good” matches/Evalues.

4. Combine GO annotations into single file.• Remove duplicates

Adding GO Annotation

• GO annotations are usually added as gene association files.• Check the number of the columns.• Can check file format against the GO guide:

• Check your analysis tool:• accepts additional GO annotations• format required

http://www.geneontology.org/GO.format.annotation.shtml

GO Enrichment tools that support agricultural species.