Transcript Curso de verao

Anotação automática de seqüências
biológicas: ontologias e sistemas de pipelines
Arthur Gruber
Instituto de Ciências Biomédicas
Universidade de São Paulo
AG-ICB-USP
Sequence annotation
•
Annotation is the process
information to a DNA sequence.
of
adding
•
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
Public databases
•
•
GenBank, EMBL and DDBJ.
All databases update each other
automatically
AG-ICB-USP
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
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
Header (EMBL)
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.
AG-ICB-USP
NCBI Header
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.
AG-ICB-USP
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
Feature keys
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
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
AG-ICB-USP
Feature qualifier
Additional information about a feature
/note="text"
/allele="text"
/number=unquoted
/citation=[number]
/codon=(seq:"text",aa:<amino_acid>)/product="text"
/protein_id="<identifier>"
/codon_start=<1
/db_xref="<database>:<identifier>" /pseudo
/standard_name="text"
/EC_number="text"
/translation="text"
/evidence=<evidence_value>
/transl_except=(pos:<base_range>,aa:<amino_acid>)
/exception="text"
/transl_table
/function="text"
/usedin=accnum:feature_label
/gene="text"
/label=feature_label
/map="text"
AG-ICB-USP
Features (EMBL)
AG-ICB-USP
Features (NCBI)
FEATURES
source
Location/Qualifiers
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
AG-ICB-USP
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
/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
/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
Naming protocols
•
Hypothetical protein
unknown function and no homology
•
Conserved hypothetical protein
unknown function WITH homology
•
Alcohol dehydrogenase like
looks a bit like it, but may not be.
•
Putative alcohol dehydrogenase
probably a alcohol dehydrogenase
•
Alcohol dehydrogenase
this has previously been
characterised and shown to be
alcohol dehydrogenase in this
organism.
AG-ICB-USP
/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
Transitive Annotation
•
•
•
AKA annotation catastrophe
Junk in = Junk out
Mis-annotations
spread
through
incorrect database submissions.
AG-ICB-USP
How can we standardize the
annotation terms?
AG-ICB-USP
Through a dynamic
controlled vocabulary
AG-ICB-USP
AG-ICB-USP
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.
Ontology Structure
cell
membrane
mitochondrial
membrane
Directed Acyclic Graph
(DAG) - multiple
parentage allowed
chloroplast
chloroplast
membrane
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
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".
nucleus
Part_of relationship
chromosome
True Path Violations
..”the pathway from a child term all the way up to its top-level parent(s) must always be true".
chromosome
Is_a relationship
Mitochondrial
chromosome
True Path Violations
..”the pathway from a child term all the way up to its top-level parent(s) must always be true".
nucleus
A mitochondrial chromosome is not part of a nucleus!
Part_of relationship
chromosome
Is_a relationship
Mitochondrial
chromosome
True Path Violations
..”the pathway from a child term all the way up to its top-level parent(s) must always be true".
nucleus
Part_of
relationship
Nuclear
chromosome
chromosome
Is_a relationship
mitochondrion
Part_of
relationship
Mitochondrial
chromosome
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)
Term-term relationship
• is_a
• The is_a relationship is a simple classsubclass relationship, where A is_a B means
that A is a subclass of B
• For example, nuclear chromosome is_a
chromosome.
GO:0043232 : intracellular non-membrane-bound organelle
GO:0005694 : chromosome
GO:0000228 : nuclear chromosome
AG-ICB-USP
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
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
AG-ICB-USP
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
AG-ICB-USP
Search result for toxin
AG-ICB-USP
Relationships in GO
•“is-a”
•“part of”
AG-ICB-USP
GO paths to terms
AG-ICB-USP
GO definitions
AG-ICB-USP
Pyruvate
dehydrogenase
AG-ICB-USP
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
GO Evidence codes
• 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
AG-ICB-USP
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
AG-ICB-USP
AG-ICB-USP
AG-ICB-USP
InterPro-to-GO
EC number-to-GO
AG-ICB-USP
SP keyword-to-GO
AG-ICB-USP
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
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
Sequence Ontology
•
How to edit an ontology file?
• OBO-Edit – an ontology editor for
biologists
• OBO-Edit compliant format
AG-ICB-USP
Generic feature format 3
•
Generic format for sequence annotation
interchange
•
•
•
Tab-delimited text file
Represents features in hierarchical view
Uses a controlled vocabulary – is
compliant to Sequence Ontology
AG-ICB-USP
Generic feature format 3
•
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"
AG-ICB-USP
Generic feature format 3
•
•
•
•
•
•
•
•
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"
How to annotate these splicing variants
using Sequence Ontology terms and the
GFF3?
• 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
##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
ctg123 . exon
ctg123 . exon
ctg123 . exon
ctg123 . exon
ctg123 . exon
1300
1050
3000
5000
7000
1500
1500
3902
5500
9000
.
.
.
.
.
+
+
+
+
+
.
.
.
.
.
ctg123 . CDS
ctg123 . CDS
ctg123 . CDS
ctg123 . CDS
1201
3000
5000
7000
1500
3902
5500
7600
.
.
.
.
+
+
+
+
0
0
0
0
ID=exon00001;Parent=mRNA00003
ID=exon00002;Parent=mRNA00001,mRNA00002
ID=exon00003;Parent=mRNA00001,mRNA00003
ID=exon00004;Parent=mRNA00001,mRNA00002,mRNA00003
ID=exon00005;Parent=mRNA00001,mRNA00002,mRNA00003
ID=cds00001;Parent=mRNA00001;Name=edenprotein.1
ID=cds00001;Parent=mRNA00001;Name=edenprotein.1
ID=cds00001;Parent=mRNA00001;Name=edenprotein.1
ID=cds00001;Parent=mRNA00001;Name=edenprotein.1
ctg123 . CDS
ctg123 . CDS
ctg123 . CDS
1201 1500 . + 0 ID=cds00002;Parent=mRNA00002;Name=edenprotein.2
5000 5500 . + 0 ID=cds00002;Parent=mRNA00002;Name=edenprotein.2
7000 7600 . + 0 ID=cds00002;Parent=mRNA00002;Name=edenprotein.2
ctg123 . CDS
ctg123 . CDS
ctg123 . CDS
3301 3902 . + 0 ID=cds00003;Parent=mRNA00003;Name=edenprotein.3
5000 5500 . + 2 ID=cds00003;Parent=mRNA00003;Name=edenprotein.3
7000 7600 . + 2 ID=cds00003;Parent=mRNA00003;Name=edenprotein.3
ctg123 . CDS
ctg123 . CDS
ctg123 . CDS
3391 3902 . + 0 ID=cds00004;Parent=mRNA00003;Name=edenprotein.4
5000 5500 . + 2 ID=cds00004;Parent=mRNA00003;Name=edenprotein.4
7000 7600 . + 2 ID=cds00004;Parent=mRNA00003;Name=edenprotein.4
Generic feature format 3
•
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
AG-ICB-USP
Testing the GFF3 Validator
AG-ICB-USP
Testing the GFF3 Validator
Let’s change the feature names
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
Annotation viewing and editing
Artemis
•
Artemis is written in Java, and is available for
UNIX, GNU/Linux, BSD, Macintosh and MSWindows systems.
•
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.
AG-ICB-USP
AG-FMVZ-USP
AG-FMVZ-USP
AG-FMVZ-USP
AG-FMVZ-USP
AG-FMVZ-USP