Advanced practical course in genome bioinformatics DAY 6: Functional annotation. Petri Törönen Earlier version Patrik Koskinen

Transcription

1 Advanced practical course in genome bioinformatics DAY 6: Functional annotation Petri Törönen Earlier version Patrik Koskinen

2 Genome project roadmap After experimental design and preparations a genome project can be roughly split into the following steps: 1. Sequencing 2. (de novo) assembly, scaffolding 3. RNA-sequencing and mapping 4. Gene prediction 5. Manual & functional annotation You are herej! 6. Submission and publication of the genome in a biodatabase 7. Further downstream analysis

3 Three sections Background Methods Demonstration of tools

4 Outline of Background Goal How function can be defined Description Gene Ontology What can be used to predict function What I am omitting here Why these two matter

5 GOAL You have an unknown protein sequence It should be functionally annotated In wet lab (precise, slow) In silico (less precise, faster) When having thousands of sequences, wet lab is not an option Collective manual in-silico annotation of sequences (hundreds of scientists) Combined use of few automated in-silico methods

6 GOAL Protein sequence: MAVQISKKRKFVADGIFKAELNEFLTRELAEDGYSGVEVRVTPTRTEIIILATRTQNVLG EKGRRIRELTAVVQKRFGFPEGSVELYAEKVATRGLCAIAQAESLRYKLLGGLAVRRACY GVLRFIMESGAKGCEVVVSGKLRGQRAKSMKFVDGLMIHSGDPVNYYVDTAVRHVLLRQG VLGIKVKIMLPWDPTGKIGPKKPLPDHVSIVEPKDEILPTTPISEQKGGKPEPPAMPQPV PTA Is this informative?

7 GOAL Is this better? (Various annotation types) 40S ribosomal protein S3 Involved in translation as a component of the 40S small ribosomal subunit (PubMed: ). Has endonuclease activity and plays a role in repair of damaged DNA (PubMed: ) GO: structural constituent of ribosome GO: damaged DNA binding

8 Examples (Good, Bad, Ugly) tartrate-resistant acid phosphatase type 5 precursor [Homo sapiens] >NP_ tartrate-resistant acid phosphatase type 5 precursor [Homo sapiens] hypothetical protein PANDA_021498, partial [Ailuropoda melanoleuca] GenBank: EFB >EFB hypothetical protein PANDA_021498, partial [Ailuropoda melanoleuca]

9 How function is defined How can we describe a function for a gene?

10 How function is defined Functional description as human readable text Linking gene to Key Words (Uniprot) Linking gene to Gene Ontology classes Linking gene to Enzyme categories Linking gene to Signalling Pathways or Biochemical Pathways (KEGG) Linking Domain to functional activity Focus on description and on Gene Ontology

11 Human readable descriptions tartrate-resistant acid phosphatase type 5 precursor [Homo sapiens] Summary: This gene encodes an iron containing glycoprotein which catalyzes the conversion of orthophosphoric monoester to alcohol and orthophosphate. It is the most basic of the acid phosphatases and is the only form not inhibited by L(+)- tartrate. [provided by RefSeq, Aug 2008].

12 Gene Ontology (GO) GO represents a popular standard currently in the gene annotation GO represents classes that represent gene function Genes in same process are grouped to same class Easy summary for genes with similar function Easier to predict than text descriptions

13 Gene Ontology (GO) 3 sub-parts: Biological Process, Molecular Function, Cellular Localization Molecular Function => chemical activity Biological Process => Biology, cellular process Cellular localization => Location of gene in cell Hierarchical structure Categories with very precise function Categories with less precise function Categories with very broad function

14 Gene Ontology tartrate-resistant acid phosphatase type 5 precursor [Homo sapiens]

15 Advantages of GO Cross species comparison Already used by several databases Comprehensive GO covers all biological and chemical processes Many terms per gene product Simplify querying Uses restricted vocabulary developed by curators and annotators Use of evidence code How reliable is the given information

16 Disadvantages of GO Class gives only an approximate description of the gene Misannotations / Unreliable annotations This problem occurs Pathways are sometimes better representations KEGG database

17 Methods outline Stupid method What can be used to predict function Current in-silico methods Method Comparisons

18 Stupid method Run BLAST search Take the first sequence hit

19 Stupid Functional annotation Traditional way to go: Nearest neighbour = query sequence Threshold in search space (e.g. Blast e-val 1e-5)

20 Example Blast results from the Meliteae Cinxia (The Glanville fritillary butterfly) Sequences producing significant alignments: AGAP PA Score (bits) E-Value tr Q7QF43 Q7QF43_ANOGA AGAP PA OS=Anopheles gambiae GN=AGA e-35 tr B4JK90 B4JK90_DROGR GH12613 OS=Drosophila grimshawi GN=GH e-31 tr Q29H35 Q29H35_DROPS GA13573 OS=Drosophila pseudoobscura pseud e-30 tr B4N1J8 B4N1J8_DROWI GK16321 OS=Drosophila willistoni GN=GK e-29 tr B4PXZ6 B4PXZ6_DROYA GE17857 OS=Drosophila yakuba GN=GE17857 P e-29 tr Q9VZ71 Q9VZ71_DROME CG15211, isoform A OS=Drosophila melanoga e-29 tr B4R7M7 B4R7M7_DROSI GD16987 OS=Drosophila simulans GN=GD e-29 tr B4IDT9 B4IDT9_DROSE GM11433 OS=Drosophila sechellia GN=GM e-29 tr B3NVE7 B3NVE7_DROER GG18369 OS=Drosophila erecta GN=GG18369 P e-29 tr B3MQD3 B3MQD3_DROAN GF20425 OS=Drosophila ananassae GN=GF e-29 tr B4L7Y1 B4L7Y1_DROMO GI11027 OS=Drosophila mojavensis GN=GI e-28 tr Q16VM8 Q16VM8_AEDAE Putative uncharacterized protein (Fragmen e-28 tr B0WJ18 B0WJ18_CULQU Putative uncharacterized protein OS=Culex e-27 tr C1C2H5 C1C2H5_9MAXI Plasmolipin OS=Caligus clemensi GN=PLLP P e-20 tr Q1DGM2 Q1DGM2_AEDAE Putative uncharacterized protein (Fragmen e-19 tr C3ZW39 C3ZW39_BRAFL Putative uncharacterized protein OS=Branc e-11 tr A3KQ86 A3KQ86_DANRE Novel protein similar to vertebrate trans e-10 tr C3YLB7 C3YLB7_BRAFL Putative uncharacterized protein OS=Branc e-10 sp Q8CJ61 CKLF4_MOUSE CKLF-like MARVEL transmembrane domain-cont e-09 tr A4IFB9 A4IFB9_BOVIN CMTM4 protein OS=Bos taurus GN=CMTM4 PE= e-09 sp P47987 PLLP_RAT Plasmolipin OS=Rattus norvegicus GN=Pllp PE= e-08 sp Q9DCU2 PLLP_MOUSE Plasmolipin OS=Mus musculus GN=Pllp PE=2 SV=1 66 1e-08 tr Q4SI17 Q4SI17_TETNG Chromosome 5 SCAF14581, whole genome shot e-08 tr B1H2E6 B1H2E6_XENTR LOC protein OS=Xenopus tropicali e-08 tr A7YYE5 A7YYE5_DANRE CKLF-like MARVEL transmembrane domain con e-08 tr C4Q9A0 C4Q9A0_SCHMA Marvel-containing potential lipid raft-as e-08 tr Q6DGM6 Q6DGM6_DANRE CKLF-like MARVEL transmembrane domain con e-08 tr C3ZW41 C3ZW41_BRAFL Putative uncharacterized protein (Fragmen e-08 tr C1BJ60 C1BJ60_OSMMO Plasmolipin OS=Osmerus mordax GN=PLLP PE= e-07 sp Q8IZR5 CKLF4_HUMAN CKLF-like MARVEL transmembrane domain-cont e-07 Is this an informative annotation to adopt?

21 Example Blast results from the Meliteae Cinxia (The Glanville fritillary butterfly) Sequences producing significant alignments: Score (bits) E-Value tr Q7QF43 Q7QF43_ANOGA AGAP PA OS=Anopheles gambiae GN=AGA e-35 tr B4JK90 B4JK90_DROGR GH12613 OS=Drosophila grimshawi GN=GH e-31 tr Q29H35 Q29H35_DROPS GA13573 OS=Drosophila pseudoobscura pseud e-30 tr B4N1J8 B4N1J8_DROWI GK16321 OS=Drosophila willistoni GN=GK e-29 tr B4PXZ6 B4PXZ6_DROYA GE17857 OS=Drosophila yakuba GN=GE17857 P e-29 tr Q9VZ71 Q9VZ71_DROME CG15211, isoform A OS=Drosophila melanoga e-29 tr B4R7M7 B4R7M7_DROSI GD16987 OS=Drosophila simulans GN=GD e-29 tr B4IDT9 B4IDT9_DROSE GM11433 OS=Drosophila sechellia GN=GM e-29 tr B3NVE7 B3NVE7_DROER GG18369 OS=Drosophila erecta GN=GG18369 P e-29 tr B3MQD3 B3MQD3_DROAN GF20425 OS=Drosophila ananassae GN=GF e-29 tr B4L7Y1 B4L7Y1_DROMO GI11027 OS=Drosophila mojavensis GN=GI e-28 tr Q16VM8 Q16VM8_AEDAE Putative uncharacterized protein (Fragmen e-28 tr B0WJ18 B0WJ18_CULQU Putative uncharacterized protein OS=Culex e-27 tr C1C2H5 C1C2H5_9MAXI Plasmolipin OS=Caligus clemensi GN=PLLP P e-20 tr Q1DGM2 Q1DGM2_AEDAE Putative uncharacterized protein (Fragmen e-19 tr C3ZW39 C3ZW39_BRAFL Putative uncharacterized protein OS=Branc e-11 tr A3KQ86 A3KQ86_DANRE Novel protein similar to vertebrate trans e-10 tr C3YLB7 C3YLB7_BRAFL Putative uncharacterized protein OS=Branc e-10 sp Q8CJ61 CKLF4_MOUSE CKLF-like MARVEL transmembrane domain-cont e-09 tr A4IFB9 A4IFB9_BOVIN CMTM4 protein OS=Bos taurus GN=CMTM4 PE= e-09 sp P47987 PLLP_RAT Plasmolipin OS=Rattus norvegicus GN=Pllp PE= e-08 sp Q9DCU2 PLLP_MOUSE Plasmolipin OS=Mus musculus GN=Pllp PE=2 SV=1 66 1e-08 tr Q4SI17 Q4SI17_TETNG Chromosome 5 SCAF14581, whole genome shot e-08 tr B1H2E6 B1H2E6_XENTR LOC protein OS=Xenopus tropicali e-08 tr A7YYE5 A7YYE5_DANRE CKLF-like MARVEL transmembrane domain con e-08 tr C4Q9A0 C4Q9A0_SCHMA Marvel-containing potential lipid raft-as e-08 tr Q6DGM6 Q6DGM6_DANRE CKLF-like MARVEL transmembrane domain con e-08 tr C3ZW41 C3ZW41_BRAFL Putative uncharacterized protein (Fragmen e-08 tr C1BJ60 C1BJ60_OSMMO Plasmolipin OS=Osmerus mordax GN=PLLP PE= e-07 sp Q8IZR5 CKLF4_HUMAN CKLF-like MARVEL transmembrane domain-cont e-07 Plasmolipins Chemokine - like factor superfamily members

22 Why traditional method is stupid? Blind copying of the nearest neighbor annotation is creating errors in database annotations

23 Errors in public databases Schnoes AM, Brown SD, Dodevski I, Babbitt PC, Annotation Error in Public Databases: Misannotation of Molecular Function in Enzyme Superfamilies. PLoS Computational Biology 5(12)

24 Errors in databases So all the annotations in the databases are not correct What can be done?

25 Rational Functional annotation Collect some features for analyzed sequence Compare these features to features in known sequences Estimate the function based on the similarity with many sequences

26 What can be used to predict function Where computer programs and researchers can get the information for the sequence?

27 Function Prediction: What can we use to predict function This lecture discusses sequence based features! Sequence homology (BLAST result list) Phylogenetic tree of sequences Protein Domains (PFAM domains) Short sequence patterns motifs Sequence features

28 Function Prediction: What I am omitting Literature search Gene expression (in tissues, developmental stages..) Protein Protein interaction Genetic interactions These are also important source. But only if they are available.

29 Why the two previous groups matter? Assume you want to predict genes that activate neuronal growth in mammalian fetus Assume you want to predict genes that bind oxygen

30 Sequence Homology Methods Do a BLAST search with a query sequence Collect GO classes for genes in the BLAST result hit Give a weight to each BLAST hit often log(e-value) Combine the scores from the genes that belong to same GO class Report the top best / significant GO classes

31 BLAST example re-visited Sequences producing significant alignments: Score (bits) E-Value tr Q7QF43 Q7QF43_ANOGA AGAP PA OS=Anopheles gambiae GN=AGA e-35 tr B4JK90 B4JK90_DROGR GH12613 OS=Drosophila grimshawi GN=GH e-31 tr Q29H35 Q29H35_DROPS GA13573 OS=Drosophila pseudoobscura pseud e-30 tr B4N1J8 B4N1J8_DROWI GK16321 OS=Drosophila willistoni GN=GK e-29 tr B4PXZ6 B4PXZ6_DROYA GE17857 OS=Drosophila yakuba GN=GE17857 P e-29 tr Q9VZ71 Q9VZ71_DROME CG15211, isoform A OS=Drosophila melanoga e-29 tr B4R7M7 B4R7M7_DROSI GD16987 OS=Drosophila simulans GN=GD e-29 tr B4IDT9 B4IDT9_DROSE GM11433 OS=Drosophila sechellia GN=GM e-29 tr B3NVE7 B3NVE7_DROER GG18369 OS=Drosophila erecta GN=GG18369 P e-29 tr B3MQD3 B3MQD3_DROAN GF20425 OS=Drosophila ananassae GN=GF e-29 tr B4L7Y1 B4L7Y1_DROMO GI11027 OS=Drosophila mojavensis GN=GI e-28 tr Q16VM8 Q16VM8_AEDAE Putative uncharacterized protein (Fragmen e-28 tr B0WJ18 B0WJ18_CULQU Putative uncharacterized protein OS=Culex e-27 tr C1C2H5 C1C2H5_9MAXI Plasmolipin OS=Caligus clemensi GN=PLLP P e-20 tr Q1DGM2 Q1DGM2_AEDAE Putative uncharacterized protein (Fragmen e-19 tr C3ZW39 C3ZW39_BRAFL Putative uncharacterized protein OS=Branc e-11 tr A3KQ86 A3KQ86_DANRE Novel protein similar to vertebrate trans e-10 tr C3YLB7 C3YLB7_BRAFL Putative uncharacterized protein OS=Branc e-10 sp Q8CJ61 CKLF4_MOUSE CKLF-like MARVEL transmembrane domain-cont e-09 tr A4IFB9 A4IFB9_BOVIN CMTM4 protein OS=Bos taurus GN=CMTM4 PE= e-09 sp P47987 PLLP_RAT Plasmolipin OS=Rattus norvegicus GN=Pllp PE= e-08 sp Q9DCU2 PLLP_MOUSE Plasmolipin OS=Mus musculus GN=Pllp PE=2 SV=1 66 1e-08 tr Q4SI17 Q4SI17_TETNG Chromosome 5 SCAF14581, whole genome shot e-08 tr B1H2E6 B1H2E6_XENTR LOC protein OS=Xenopus tropicali e-08 tr A7YYE5 A7YYE5_DANRE CKLF-like MARVEL transmembrane domain con e-08 tr C4Q9A0 C4Q9A0_SCHMA Marvel-containing potential lipid raft-as e-08 tr Q6DGM6 Q6DGM6_DANRE CKLF-like MARVEL transmembrane domain con e-08 tr C3ZW41 C3ZW41_BRAFL Putative uncharacterized protein (Fragmen e-08 tr C1BJ60 C1BJ60_OSMMO Plasmolipin OS=Osmerus mordax GN=PLLP PE= e-07 sp Q8IZR5 CKLF4_HUMAN CKLF-like MARVEL transmembrane domain-cont e-07 Plasmolipins Chemokine - like factor superfamily members

32 Sequence Homology Methods Simple method Can fail to detect some similarities Programs BLAST2GO ( GOTCHA ( ARGOT 2 ( PFP ( PANNZER (

33 Phylogenetic tree methods Create the pair-wise distances for the set of genes Do a hierarchical clustering of genes Map the know GO functions to cluster tree Look for unknown genes in a cluster with many genes from the same GO class Report the top best / significant GO classes More =>

34 Phylogenetic tree methods These should outperform sequence homology methods Require a set of related genes Often much heavier calculations Programs: SIFTER (

35 Prediction with Protein domains Look what protein domains there are in query protein (PFAM) Map the functions that are linked to domains to your query sequence PFAM2GO Programs: InterProScan + PFAM2GO Drawbacks: This mapping is same in plant, mammal, bacteria Many domains to specific function

36 Prediction with Protein domains Benefits: Can create annotation from separate domains Similar seq:s do not have to be in database Programs: InterProScan ( Drawbacks: The mapping is same in plant, mammal, bacteria Many domains to specific function

37 Our contribution: PANNZER Use BLAST result list Add Taxonomic information Score GO classes using a score that takes the frequency of GO class in seq. DB into account Method is used to predict: GO Classes Description line

38 Our contribution: PANNZER Benefits: Taking the species taxonomy into account Improved use of statistics Drawbacks: Use (currently) only sequence similarity

39 Method comparisons Many annotation methods available All methods claim to be best available What methods are really the best?

40 Critical Assessment of Function Annotations (CAFA) Select a set of unknown genes Ask research groups to predict GO terms After dead line start collecting new annotations for genes Next evaluate the methods

41 Critical Assessment of Function Annotations (CAFA) Radivojac et al. A large-scale evaluation of computational protein function prediction. Nature Methods Jan 27. doi: /nmeth.2340.

42 CAFA 1 Most successful methods JonesUCL Argot 2 Pannzer

43 Method demonstrations InterProScan Argot Pannzer

44 Demo sequences 5 sequences 3 from eukaryota, 2 in prokaryota All are annotated as unknown in DB They can be still annotated based on the sequence Sequences are here: /Exercise_material_day7.txt

45 InterProScan InterProScan is metaserver that looks many sequence features Many of these features can be used to annotate sequences You have to give one sequence at the time Results include detailed description of domain functions

46 InterProScan

47 Results S es 5-S es 5-S oy 5-S es 5-S es

48 Argot2 Tool that processes BLAST output One of the best methods in CAFA 1

49 Argot2 Results: uences.php?js=13038

50 PANNZER2 Processes results similar to BLAST Predicts text and Gene Ontology classes Significantly faster than the other tools

51 Conclusion These methods increasingly needed Some methods exist Unfortunately no clear evaluation Remember: These are predictions. No certain info until they are tested in wet lab