1. Anotação automática de seqüências biológicas: ontologias e sistemas de pipelines Arthur Gruber Instituto de Ciências Biomédicas Universidade de São Paulo AG-ICB-USP

2. Sequence annotation Annotation is the process of adding information to a DNA sequence. The information usually has DNA coordinate. Features could be repeats, genes, promoters, protein domains…….. Features can be linked to other databases e.g. Pfam/Pubmed AG-ICB-USP

3. Public databases GenBank, EMBL and DDBJ. All databases update each other automatically AG-ICB-USP

4. Feature table http://www.ncbi.nlm.nih.gov/projects/collab/FT/ Format definition Covers DDBJ/EMBL/GenBank Defines all accepted annotation terms and hierarchy AG-ICB-USP

5. Annotation file Contains: A header with: Information about the sequence Organism Authors References Comments A feature table containing Sequence features and co-ordinates AG-ICB-USP

6. ID PFMAL1P4 standard; DNA; INV; 66441 BP. XX AC AL031747; XX SV AL031747.8 XX DT 24-SEP-1998 (Rel. 57, Created) DT 27-APR-2000 (Rel. 63, Last updated, Version 13) XX DE Plasmodium falciparum DNA from MAL1P4 XX KW HTG; rifin; telomere; var; var-like hypothetical protein. XX OS Plasmodium falciparum (malaria parasite P. falciparum) OC Eukaryota; Alveolata; Apicomplexa; Haemosporida; Plasmodium. XX RN [1] RA Oliver K., Bowman S., Churcher C., Harris B., Harris D., Lawson D., RA Quail M., Rajandream M., Barrell B.; RT ; RL Submitted (24-SEP-1998) to the EMBL/GenBank/DDBJ databases. RL P.falciparum Genome Sequencing Consortium, The Sanger Centre, Wellcome RL Trust Genome Campus, Hinxton, Cambridge CB10 1S. Header (EMBL) AG-ICB-USP

7. LOCUS PFMAL1P4 66442 bp DNA linear INV 02-DEC-2004 DEFINITION Plasmodium falciparum DNA from MAL1P4, complete sequence. ACCESSION AL031747 AL844501 VERSION AL031747.9 GI:23477012 KEYWORDS HTG; rifin; telomere; var; var-like hypothetical protein. SOURCE Plasmodium falciparum 3D7 ORGANISM Plasmodium falciparum 3D7 Eukaryota; Alveolata; Apicomplexa; Haemosporida; Plasmodium. REFERENCE 1 AUTHORS Hall,N., Pain,A., Berriman,M., Churcher,C., Harris,B., Harris,D., TITLE Sequence of Plasmodium falciparum chromosomes 1, 3-9 and 13 JOURNAL Nature 419 (6906), 527-531 (2002) PUBMED 12368867 REFERENCE 2 AUTHORS Oliver,K., Pain,A., Berriman,M., Bowman,S., Churcher,C., Harris,B., Harris,D., Lawson,D., Quail,M., Rajandream,M., Hall,N. and Barrell,B. TITLE Direct Submission JOURNAL Submitted (24-SEP-1998) P.falciparum Genome Sequencing Consortium, The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK COMMENT On Oct 2, 2002 this sequence version replaced gi:7670004. For more information about this sequence or the Malaria Project, see http://www.sanger.ac.uk/Projects/P_falciparum. NCBI Header AG-ICB-USP

8. Feature Region of DNA that was annotated with a key/qualifier Keys: CDS, intron, miscellaneous, etc. Qualifier: notes or extra-information about a feature i.e. exon (key) /gene=“adh” (qualifier) AG-ICB-USP

9. Feature keys misc_difference misc_feature misc_recomb misc_RNA misc_signal misc_structure modified_base mRNA N_region old_sequence polyA_signal polyA_site precursor_RNA prim_transcript primer_bind promoter protein_bind RBS repeat_region repeat_unit rep_origin rRNA S_region satellite scRNA sig_peptide snRNA snoRNA source stem_loop STS TATA_signal terminator transit_peptide tRNA unsure V_region V_segment variation 3'clip 3'UTR 5'clip 5'UTR -10_signal -35_signal attenuator C_region CAAT_signal CDS conflict D-loop D_segment enhancer exon GC_signal gene iDNA intron J_segment LTR mat_peptide misc_binding AG-ICB-USP

10. Feature qualifier Additional information about a feature /allele="text" /citation=[number] /codon=(seq:"text",aa: ) /codon_start= : " /EC_number="text" /evidence= /exception="text" /function="text" /gene="text" /label=feature_label /map="text" /note="text" /number=unquoted /product="text" /protein_id=" " /pseudo /standard_name="text" /translation="text" /transl_except=(pos:,aa: ) /transl_table /usedin=accnum:feature_label AG-ICB-USP

11. Features (EMBL) AG-ICB-USP

12. Features (NCBI) AG-ICB-USP FEATURES Location/Qualifiers source 1..66442 /organism="Plasmodium falciparum 3D7" /mol_type="genomic DNA" /isolate="3D7" /db_xref="taxon:36329" /chromosome="1" repeat_region 1..583 /note="telomeric repeat" repeat_region 584..1641 /note="14bp repeat" gene join(29733..34985,36111..37349) /gene="MAL1P4.01" /note="synonyms: PFA0005w, VAR" CDS join(29733..34985,36111..37349) /gene="MAL1P4.01" /note="Subtelomeric var gene Pfam hit to PF03011 Similar to Plasmodium falciparum VaR, mal1p4.01 vaR SWALL:Q9NFB6 (EMBL:AL031747) (2163 aa) fasta scores: E(): 0, 100% id in 2163 aa" /codon_start=1 /product="erythrocyte membrane protein 1 (PfEMP1)" /protein_id="CAB89209.1" /db_xref="GI:7670005" /db_xref="GOA:Q9NFB6" /db_xref="UniProtKB/TrEMBL:Q9NFB6" /translation="MVTQSSGGGAAGSSGEEDAKHVLDEFGQQVYNEKVEKYANSKIY KEALKGDLSQASILSELAGTYKPCALEYEYYKHTNGGGKGKRYPCTELGEKVEPRFSDTLGGQCTNK KIEGNKYIKGKDVGACAPYRRLHLCSHNLESIQ

13. CDS features CDS stands for coding sequence and is used to denote genes and pseudogenes. These features are automatically translated on submission and the protein added to the protein databases. AG-ICB-USP

14. /note Note field contains all the evidence for a gene call……..plus anything else. Similarity (fasta or blast) Domain/motif information (Pfam, TMHMM, etc.) Unusual features (repeats, aa richness) AG-ICB-USP

15. /product The name of the gene product eg. Alcohol dehydrogenase Unless there is proof we must qualify... Putative Possible Always be conservative!… eg. Putative dehydrogenase dehyrogenase like protein Only piece of annotation added to the protein databases. AG-ICB-USP

16. Naming protocols Hypothetical proteinunknown function and no homology Conserved hypothetical proteinunknown function WITH homology Alcohol dehydrogenase likelooks a bit like it, but may not be. Putative alcohol dehydrogenaseprobably a alcohol dehydrogenase Alcohol dehydrogenasethis has previously been characterised and shown to be alcohol dehydrogenase in this organism. AG-ICB-USP

17. /gene The gene name eg ADH1 Only transfer a gene name if it is meaningful Never transfer a gene name like PfB0024. Is it a gene family? make sure two genes have the same name. AG-ICB-USP

18. Transitive Annotation AKA annotation catastrophe Junk in = Junk out Mis-annotations spread through incorrect database submissions. AG-ICB-USP

19. How can we standardize the annotation terms? AG-ICB-USP

20. Through a dynamic controlled vocabulary AG-ICB-USP

22. So what does that mean? From a practical view, ontology is the representation of something we know about. “Ontologies" consist of a representation of things, that are detectable or directly observable, and the relationships between those things.

23. Ontology Structure cell membrane chloroplast mitochondrial chloroplast membrane Directed Acyclic Graph (DAG) - multiple parentage allowed

24. GO topology The ontologies are structured as directed acyclic graphs Similar to hierarchies but differ in that a more specialized term (child) can be related to more than one less specialized term (parent). For example, hexose biosynthetic process has two parents, hexose metabolic process and monosaccharide biosynthetic process. AG-ICB-USP

25. True Path Violations Create Incorrect Definitions..”the pathway from a child term all the way up to its top-level parent(s) must always be true". chromosome Part_of relationship nucleus

26. True Path Violations..”the pathway from a child term all the way up to its top-level parent(s) must always be true". chromosome Mitochondrial chromosome Is_a relationship

27. True Path Violations..”the pathway from a child term all the way up to its top-level parent(s) must always be true". chromosome Mitochondrial chromosome Is_a relationship Part_of relationship nucleus A mitochondrial chromosome is not part of a nucleus!

28. True Path Violations..”the pathway from a child term all the way up to its top-level parent(s) must always be true". nucleuschromosome Nuclear chromosome Mitochondrial chromosome Is_a relationship Part_of relationship mitochondrion Part_of relationship

29. GO Definitions: Each GO term has 2 Definitions A definition written by a biologist: necessary & sufficient conditions written definition (not computable) Graph structure: necessary conditions formal (computable)

30. Term-term relationship is_a The is_a relationship is a simple class- subclass relationship, where A is_a B means that A is a subclass of B For example, nuclear chromosome is_a chromosome. AG-ICB-USP GO:0043232 : intracellular non-membrane-bound organelle GO:0005694 : chromosome GO:0000228 : nuclear chromosome

31. Term-term relationship part_of C part_of D means that whenever C is present, it is always a part of D, but C does not always have to be present For example, periplasmic flagellum part_of periplasmic space AG-ICB-USP GO:0044464 : cell part GO:0042995 : cell projection GO:0019861 : flagellum GO:0009288 : flagellin-based flagellum GO:0055040 : periplasmic flagellum GO:0042597 : periplasmic space GO:0055040 : periplasmic flagellum

32. Current Ontologies Molecular function: tasks performed by gene product Biological process: broad biological goals accomplished by ordered assemblies of molecular functions Cellular component: subcellular structures, locations and macromolecular complexes AG-ICB-USP

34. Search result for toxin AG-ICB-USP

35. Relationships in GO “is-a” “part of” AG-ICB-USP

36. GO paths to terms AG-ICB-USP

37. GO definitions AG-ICB-USP

38. Pyruvate dehydrogenase AG-ICB-USP

39. Why the interest in GO? ● Universal ontology ● Functional classification scheme with many different levels in a DAG ● Widespread interest from scientific community ● Already mappings to SP keywords and gene products-annotation on some organisms AG-ICB-USP

40. GO Evidence codes AG-ICB-USP Experimental Evidence Codes 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 Computational Analysis Evidence Codes 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 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

41. Current Mappings to GO Consortium mappings -MGD, SGD, FlyBase Swiss-Prot keywords EC numbers InterPro entries Medline ID Commercial companies -CompuGen, Proteome AG-ICB-USP

45. InterPro-to-GO

46. EC number-to-GO AG-ICB-USP

47. SP keyword-to-GO AG-ICB-USP

48. GO doesn’t cover… Gene products: e.g. cytochrome c is not in the ontologies, but attributes of cytochrome c, such as oxidoreductase activity, are. Processes, functions or components that are unique to mutants or diseases: e.g. oncogenesis is not a valid GO term because causing cancer is not the normal function of any gene. Attributes of sequence such as intron/exon parameters: these are not attributes of gene products and will be described in a separate sequence ontology (see Sequence Ontology). Protein domains or structural features. Protein-protein interactions. Environment, evolution and expression. Anatomical or histological features above the level of cellular components, including cell types. AG-ICB-USP

50. Sequence Ontology The four major aspects of the complete Sequence Ontology are: located sequence features for objects that can be located on sequence in coordinates, sequence attributes for describing the properties of features, consequences of mutation for the annotation of the effects of a mutation chromosome variation to describe large scale variations AG-ICB-USP

51. Sequence Ontology AG-ICB-USP How to edit an ontology file? OBO-Edit – an ontology editor for biologists OBO-Edit compliant format

53. Generic feature format 3 AG-ICB-USP Generic format for sequence annotation interchange Tab-delimited text file Represents features in hierarchical view Uses a controlled vocabulary – is compliant to Sequence Ontology

54. AG-ICB-USP The tab-delimited file presents 9 columns: Column 1: "seqid" Column 2: "source" Column 3: "type" Columns 4 & 5: "start" and "end" Column 6: "score" Column 7: "strand" The strand of the feature. + for positive strand (relative to the landmark), - for minus strand Column 8: "phase" Column 9: "attributes" Generic feature format 3

55. Column 1: "seqid" Column 2: "source" Column 3: "type" Columns 4 & 5: "start" and "end" Column 6: "score" Column 7: "strand" Column 8: "phase" Column 9: "attributes"

56. How to annotate these splicing variants using Sequence Ontology terms and the GFF3?

57. The annotated genome region is named “ctg123” A gene named EDEN extends from coordinates 1 to 9000 The gene encodes three alternatively-spliced variants: EDEN.1, EDEN.2 and EDEN.3 Transcript EDEN.3 presents two alternative translation start points There is a transcriptional factor binding site (a promoter) located 50 bp upstream of the translational start site of EDEN.1

58. ##gff-version 3 ##sequence-region ctg123 1 1497228 ctg123. gene 1000 9000. +. ID=gene00001;Name=EDEN ctg123. TF_binding_site 1000 1012. +. ID=tfbs00001;Parent=gene00001 ctg123. mRNA 1050 9000. +. ID=mRNA00001;Parent=gene00001;Name=EDEN.1 ctg123. mRNA 1050 9000. +. ID=mRNA00002;Parent=gene00001;Name=EDEN.2 ctg123. mRNA 1300 9000. +. ID=mRNA00003;Parent=gene00001;Name=EDEN.3

59. ctg123. exon 1300 1500. +. ID=exon00001;Parent=mRNA00003 ctg123. exon 1050 1500. +. ID=exon00002;Parent=mRNA00001,mRNA00002 ctg123. exon 3000 3902. +. ID=exon00003;Parent=mRNA00001,mRNA00003 ctg123. exon 5000 5500. +. ID=exon00004;Parent=mRNA00001,mRNA00002,mRNA00003 ctg123. exon 7000 9000. +. ID=exon00005;Parent=mRNA00001,mRNA00002,mRNA00003 ctg123. CDS 1201 1500. + 0 ID=cds00001;Parent=mRNA00001;Name=edenprotein.1 ctg123. CDS 3000 3902. + 0 ID=cds00001;Parent=mRNA00001;Name=edenprotein.1 ctg123. CDS 5000 5500. + 0 ID=cds00001;Parent=mRNA00001;Name=edenprotein.1 ctg123. CDS 7000 7600. + 0 ID=cds00001;Parent=mRNA00001;Name=edenprotein.1

60. ctg123. CDS 1201 1500. + 0 ID=cds00002;Parent=mRNA00002;Name=edenprotein.2 ctg123. CDS 5000 5500. + 0 ID=cds00002;Parent=mRNA00002;Name=edenprotein.2 ctg123. CDS 7000 7600. + 0 ID=cds00002;Parent=mRNA00002;Name=edenprotein.2 ctg123. CDS 3301 3902. + 0 ID=cds00003;Parent=mRNA00003;Name=edenprotein.3 ctg123. CDS 5000 5500. + 2 ID=cds00003;Parent=mRNA00003;Name=edenprotein.3 ctg123. CDS 7000 7600. + 2 ID=cds00003;Parent=mRNA00003;Name=edenprotein.3 ctg123. CDS 3391 3902. + 0 ID=cds00004;Parent=mRNA00003;Name=edenprotein.4 ctg123. CDS 5000 5500. + 2 ID=cds00004;Parent=mRNA00003;Name=edenprotein.4 ctg123. CDS 7000 7600. + 2 ID=cds00004;Parent=mRNA00003;Name=edenprotein.4

61. AG-ICB-USP If you writes a GFF file, you can test it! There is an online validator: http://dev.wormbase.org/db/validate_gff3/validate_gff3_online Generic feature format 3

62. Testing the GFF3 Validator

63. AG-ICB-USP

66. Let’s change the feature names

70. Annotation viewing and editing Artemis Artemis is a free genome viewer and annotation tool developed by Kim Rutherford (Sanger Institute, UK). It allows for visualization of sequence features and results of analyses, in the context of the sequence and its six-frame translation. AG-ICB-USP

71. Annotation viewing and editing Artemis Artemis is written in Java, and is available for UNIX, GNU/Linux, BSD, Macintosh and MS- Windows systems.Java It can read complete EMBL and GENBANK database entries or sequence in FASTA or raw format. Extra sequence features can be in EMBL, GENBANK or GFF format.EMBLGENBANKGFF AG-ICB-USP

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