Lecture 2. The Blast2GO annotation framework

Transcription

1 Lecture 2 The Blast2GO annotation framework Annotation steps Modulation of annotation intensity Export/Import Functions Sequence Selection Additional Tools

2 Functional assignment Annotation Transference Empirical Literature reference Filogeny Molecular interactions Gene/protein expression Biochemical assay Structure Comparison Identification of folds Sequence analysis Sequence homology Motif identification

3 Blast2GO site

4 Start Blast2GO

5 Start Blast2GO

6 Input data (in FASTA format, AA or nt) >my_favourite_species_seq1 still unknown gtgatggaaaagaaaagttttgttatcgtcgacgcatatgggtttctttttcgcgcgtattatgcgctgcctggattaagcacctcatacaattttcctgtaggaggtgtatatggtttt ataaacatacttttgaaacatctctctttccacgatgcagattatttagttgtggtatttgattcggggtcgaaaaattttcgtcacactatgtattccgaatacaaaactaatcgccc taaagcaccagaggatctgtcactacaatgtgctccgctacgtgaggctgttgaagcgtttaatattgtaagtgaagaagtgcttaactacgaagcagacgacgtaatagct acactctgtacaaaatatgcatctagtaatgttggagtgagaatactgtcagcagataaggatttactacaactcctaaatgataatgttcaagtttacgaccctataaaaagc agatacctcaccaatgaatacgttttagaaaaatttggtgtttcatcagataagttgcatattgatacggttgcatcgagttataatgagaaaattattctcagctaagctgtacac cgtttattacacactcgaaaggccgttag as >my_favourite_species_seq2 no clue df ttgttagctaaaaaggaagactttcacacctttggtaatggtgttggctctgctggaacaggtggagttgtagtttctgcatccatgttgtctgcggatttttcaaatcttagagaag agatagcagcggttagtacggctggtgcagattggttacacattgatgtgatggatgggtgcttcgtccccagtttgactatgggtcctgtggtgatttccggcattaggaaatgt acaaatatgtttcttgatgtgcatttgatgattaatcgcccaggcgatcatctgaagagtgtggtagatgctggagctgataagatagagcacattcgcaagatgatagagga asdf aagctcatcaaccgcgaaaatcgctgttgatggtggtgtttcaacggataatgcccgggctgttatcgaggcaggtgcgaatatactcgttgttggaacggcgctgtttgctgc tgacgatatgagtaaagttgtaagaactttaaaatcattttaa >my_favourite_species_seq3 just sequenced gtgggactgctcatccctgtaggcagggtggctattttttgtgtaaaggcagtctttcatagtcttgtaccgccatactatctatggataactacaaagcagttttttgaggtgtggtt tttctctcttcctatagtagcagttacatctttgtttacgggaggcgcgttagcccttcaggataccctcgtgggaagcgctaaagtatcagggtaatggagtttttactcctgcaa gatgtaatagagggtctggtaaaagctgtatcgtttgggctggtaatttcgctagttgggtgttacaacgggtatcactgtgagataggcgcaaggggtgtaggaacagcga caacaaaaacttcggtagcagcttctatgctcataattttgttaaactatataattactgttttttacgcgta >my_favourite_species_seq4 we will see soon... atgtacgctgtatctctttcaaatttgcatgtctctttcaacaacaaggaggttttgaaaggtgttgacttggacatagcatggggggattccctggttatactgggagaatctggt agtggaaagtctgtactaacaaaggttgtattgggtctaatagtgccccaagagggaagtgttactgtagatggcaccaatattcttgagaataggcagggcatcaagaatt ttagtgttttgtttcaaaactgtgcgttatttgacagtcttacgatttgggaaaatgtagtattcaatttccgtaggaggcttcgtttagataaggataatgccaaggctttggctttac ggggattggagcttgtgggattggacgccagtgtaatgaacgtgtatcctgtggagctatcaggcgggatgaaaaagcgcgtagctttggcaagagctattataggtagtcc caaaattctaattttggatgagccaacttcgggattggatcctataatgtcttcagtggt

7 Blast2GO PRO Your Login: b2goeiras Your Password: b2goeiras2012

8 Blast2GO Application (1) Blast (2) Mapping (3) Annotation Main Sequence Table Any operation will only affect to selected sequences!!!! Application statistics Blast results Application messages Graph visualisation

9 The First Check Click on the green arrow to check you can connect to DB A GO graph should appear

10 Load Sequences

11 Run BLAST search BLAST against NCBI or locally Choose different DBs In combination with URL Limit to query-hit overlap Recommended to save as XML Text mining on BLAST hit description

12 Choose other DB at NCBI Set at blast2go.properties file

13 BLAST Results RED

14 Blast Distribution Charts Evaluate the similarity of your sequences with public DBs

15 Single Sequence Menu Single Sequence Menu

16 Mapping Results GREEN

17 Resources for mapping Gene Ontology Database NCBI data-files: gene2accession ( entries) gene_info ( entries) Protein Information Resource (PIR): Non-Redundant Reference Protein Database including PSD, UniProt, Swiss-Prot, TrEMBL, RefSeq, GenPept and PDB BLAST Hit IDs ACCs/GIs Gene-Symbols EC Mapping Resources GO terms sim %

18 Annotation Menu BLAST based annotation Other Annotation modes Validation and Annex

19 Annotation Allows to set a minimum percentage of the HIT sequence which should be expand by the QUERY sequence This helps to avoid the problem of cis-annotation

20 Annotation Result BLUE

21 Graph Visualization Root GO term Intermediate GO term Source/Hit GO term Annotated GO term

22 Annotation Charts

23 Annotation Charts Commonly, level 5 is the most abundant specificity level in the Gene Ontology

24 Additional Annotation: ANNEX Recovers implicit biological process and cellular component GO terms based on molecular function annotations Molecular Function is involved in Biological Process Myhre et al, Bioinformatics 2006 acts in Cellular Component

25 Additional Annotation: InterProScan Runs InterProScan searches at the EBI through Blast2GO Once you have completed your InterPro annotation, results can be transformed to GO terms and merged to Blast annotation Results are stored at your computer as XML files. You can upload them later

26 InterProScan Results Column with InterProScan results

27 Additional Annotation: GOSlim GOSlim is a reduction of the Gene Ontology to a more reduced vocabulary Helps to summarize information After GOSlim transformation sequences get YELLOW Different GOSlims available at Blast2GO

28 Enzyme annotation and Kegg Maps GO Enzyme Codes KEGG maps

29 Additional Annotation: Manual Curation You can modify manually annotation of particular sequences If you click in this box, curated sequences get purple

30 Export Results Saves the complete B2G project (heavy) Export annotation results in different formats

31 Export formats.annot C04018C10 C04018C10 C04018A12 C04018A12 GO: EC: GO: GO: mitogen-activated protein kinase 3 Also for import! class iv chitinase GeneSpring Format C04013E10 response to water deprivation; regulation ofnucleus; transcription; multicellular organismal transcription development; factorresponse activity; to abscisic acid stimulus; C04013A12 translation; ribosome; plastid; structural constituent of ribosome; C04013C12 galactose metabolic process; plastid; aldose 1-epimerase activity; carbohydrate binding; GoStat C04018C10 C04018A12 C04018C ,9409,6979,10200,5524, ,272, ,12505,8233 By Seq C04018A02 C04018C02 C04018G02 glyoxalase i metallothionein-like protein protein phosphatase GO: GO: GO: F:lactoylglutathione lyase activity F:metal ion binding C:protein serine/threonine phosphatase complex

32 More export formats Export Sequence Table Seq. Name C04018C12 C04018E12 C04018G12 C04018A02 C04018C02 C04018E02 C04018G02 C04018C04 C04018E04 C04018G04 C04018A06 Seq. Description Seq. Length #Hits min. evaluemean Similarity#GOs GOs Enzyme Codes InterProScan cysteine proteinase inhibitor % 3 F:GO: ; C:GO: ; F:GO: IPR000010; IPR protein phosphatase 2c % 2 N:GO: ; F:GO: IPR001932; IPR alpha beta fold family protein % 4 F:GO: ; C:GO: ; C:GO: ; noipr P:GO:00 glyoxalase i % 2 P:GO: ; F:GO: EC: IPR004360; noipr metallothionein-like protein % 1 F:GO: IPR haemolysin-iii related familyexpressed % 1 C:GO: noipr protein phosphataseexpressed % 5 C:GO: ; N:GO: ; P:GO: ; no IPS match C:GO:00 phosphoglycerate bisphosphoglycerate mutase 780 family 20 protein % 2 P:GO: ; F:GO: IPR001345; IPR polyubiquitin % 2 P:GO: ; C:GO: IPR000626; IPR meiotic recombination % 21 C:GO: ; P:GO: ; F:GO: ; IPR003701; IPR F:GO:000 late embryogenesis-abundant protein % 2 P:GO: ; P:GO: no IPS match Export BestHit Data Sequence name C04018C10 C04018E10 C04018G10 C04018A12 C04018C12 C04018E12 C04018G12 C04018A02 C04018C02 Sequence desc. Sequence lengthhit desc. Hit ACC E-Value Similarity Score Alignment lengthpositives mitogen-activated protein kinase gi gb ABM mitogen-activated ABM E-123 protein kinase99 [Citrus sinensis] NA--706 gi emb CAO unnamed CAO62459 protein 2.69E-036 product [Vitis83 vinifera] protein 620 gi gb ABI ABI52743 kda putative 7.47E-015 secreted protein 63 [Argas monolakensis] class iv chitinase 715 gi gb AAC chitinase AAC35981 CHI1 1.45E-061 [Citrus sinensis] cysteine proteinase inhibitor 663 gi gb AAF AF265551_1cysteine AAF E-025 protease inhibitor 83 [Manihot esculenta] protein phosphatase 2c 663 gi gb AAS protein AAS86762 phosphatase 2.76E-077 2C [Lycopersicon esculentum] alpha beta fold family protein 578 gi emb CAN hypothetical CAN E-084 protein [Vitis vinifera] >gi emb CAO unnam glyoxalase i 600 gi emb CAB hypothetical CAB09799 protein 2.16E-064 [Citrus x paradisi] metallothionein-like protein 625 gi dbj BAA metallothionein-like BAA E-014 protein [Citrus100 unshiu]

33 Sequence Selection Sequence Selection tool to obtain a selection based on annotation status

34 Sequence Selection By Name/Description By Function

35 View Menu Functions to switch between displaying IDs or descriptions for GO annotation or InterPro results

36 Other Tools Permits to reduce the project size Manipulation of sequence desc. Merging.annot and.dat projects Get more out of your memory Check when connection problems