Functional annotation Datasources Konstantinos Mavrommatis Kmavrommatis@lbl

MGM workshop. 19 Oct 2010

Functional annotationFunctional annotationDatasourcesDatasources

Konstantinos MavrommatisKonstantinos Mavrommatis

[email protected]@lbl.gov


Let’s get started… Let’s get started…

Tools & Information from databases are used to predict the function of a protein (functional annotation).Product nameEnzyme catalog numberDomain architecture


But what is function?But what is function?

cobalamin biosynthetic enzyme, cobalt-precorrin-4 methyltransferase (CbiF)

molecular/enzymatic (methyltransferase) Reaction (methylation)

Substrate (cobalt-precorrin-4)

Ligand (S-adenosyl-L-methionine)

metabolic (cobalamin biosynthesis)

physiological (maintenance of healthy nerve and red blood cells, through B12).


Functional annotationFunctional annotation

Predict the biochemistry and physiology of an organism based on its genome sequence

Explain known biochemical and physiological properties


Function predictionFunction prediction

Function transfer by homology

Homology implies a common

evolutionary origin.

not retention of similarity in any of their properties.

Homology ≠ similarity of function.

Punta & Ofran. PLOS Comp Biol. 2008


Trust transfer of Trust transfer of annotation ?annotation ?

Punta & Ofran. PLOS Comp Biol. 2008


Dos and Don’tsDos and Don’ts

Type Don’t Do

Homology Same function Probability for same function

Orthology Same function Probability for same function

Paralogy Same function Probability for same function

Sequence similarity Same function Probability for same function

High sequence similarity Same function Probability for same function

Same sequence Same function Probability for same function


What if nothing is similar ? What if nothing is similar ?

Subcellular localization

Gene contextSpecial featuresPrediction of

binding residues (DISIS, bindN)

Cytoplasm

S ~ S S ~ S

Periplasm


Genome annotation

Model pathway

Annotation should make Annotation should make sensesense

SubstrateA

SubstrateB

SubstrateC

SubstrateDEnzyme 2Enzyme 1 Enzyme 3

Enzyme 2? ?Enzyme 1 Enzyme 3 ✓


Annotation should make senseAnnotation should make sense


DatabasesDatabases

Databases used for the analysis of biological molecules.

Databases contain information organized in a way that allows users/researchers to retrieve and exploit it.

Why bother?Store information.Organize data.Predict features (genes, functions ...).Predict the functional role of a feature (annotation).Understand relationships (metabolic reconstruction).


Primary nucleotide Primary nucleotide databasesdatabases

EMBL/GenBank/DDBJ EMBL/GenBank/DDBJ ((http://

www.ncbi.nlm.nih.gov/,http://www.ebi.ac.uk/embl,http://www.ebi.ac.uk/embl))

Archive containing all sequences from: genome projects sequencing centers individual scientists patent offices

The sequences are exchanged between the three centers on a daily basis.

Database is doubling every 10 months.

Sequences from >140,000 different species.

1400 new species added every month.

Year Base pairs Sequences2004 44,575,745,17640,604,3192005 56,037,734,46252,016,7622006 69,019,290,70564,893,7472007 83,874,179,73080,388,3822008 99,116,431,94298,868,465


Primary protein sequence Primary protein sequence databasesdatabases

Contain coding sequences derived from the translation of nucleotide sequences GenBank

Valid translations (CDS) from nt GenBank entries.

UniProtKB/TrEMBL (1996) Automatic CDS translations from

EMBL. TrEMBL Release 40.3 (26-May-2009)

contains 7,916,844 entries.


Errors in Errors in databasesdatabases

There are a lot of errors in the primary sequence databases: In the sequences themselves:

Sequencing errors.Cloning vectors sequences.

For the annotations, the free submission of entries results to: Inaccuracies, omissions, and even mistakes. Inconsistencies between some fields.


RedundancyRedundancy

Redundancy is a major problem.

Entries are partially or entirely duplicated:

e.g. 20% of vertebrate sequences in GenBank.

{ {

{

Partial and completesequence duplications


NCBI Derivative Sequence NCBI Derivative Sequence DataData

ATTGACTA

TTGACA

CGTGAAT

TGACTA

TATAGCCG

ACGTGC

ACGTGCACGTGC

TTGACA

TTGACA

TTGACA

CGTGA

CGTGA

CGTGA

ATTGACTA

ATTGACTAATTGACTA

ATTGACTA

TATAGCCG

TATAGCCG

TATAGCCG

TATAGCCG

GenBank

TATAGCCG TATAGCCGTATAGCCGTATAGCCG

ATGA

CATT

GAGA

ATT

ATTCC GAGA

ATTCCGAGA

ATTC GAGA

ATTC

GAGA

ATTCC GAGA

ATTCC

UniGene

RefSeq

GenomeAssembly

Labs

Curators

Algorithms

TATAGCCGAGCTCCGATACCGATGACAA


RefSeqRefSeq

Curated transcripts and proteins. reviewed by NCBI staff.

Model transcripts and proteins. generated by computer algorithms.

Assembled Genomic Regions (contigs).Chromosome records.


Secondary protein Secondary protein databasesdatabases

Uniprot/SWISS-PROT (1986) (http://ca.expasy.org/spro) a curated protein sequence database

high level of annotation (such as the description of the function of a protein, its domains structure, post-translational modifications, variants, etc.)

a minimal level of redundancy

high level of integration with other databases


Classification databasesClassification databases

Groups (families/clusters) of proteins based on… Overall sequence similarity. Local sequence similarity. Presence / absence of specific features (active site, signal

peptides… ). Structural similarity. ...

These groups contain proteins with similar properties. Specific function, enzymatic activity. General function. Evolutionary relationship. …


Overall sequence Overall sequence similaritysimilarity


Clusters of orthologous groups Clusters of orthologous groups (COGs)(COGs)

COGs were delineated by comparing protein sequences encoded in 43 complete genomes representing 30 major phylogenetic lineages. Each Cluster has representatives of at least 3 lineages

A function (specific or broad) has been assigned to each COG.

http://www.ncbi.nlm.nih.gov/COG/


How it worksHow it works

Reciprocal best hitBidirectional best hit

Blast best hitUnidirectional best hit

COG1COG2


Profiles & PfamProfiles & Pfam

A method for classifying proteins into groups exploits region similarities, which contain valuable information (domains/profiles).

These domains/profiles can be used to detect distant relationships, where only few residues are conserved.


Regions similarityRegions similarity


http://pfam.sanger.ac.uk

HMMs of protein alignments(local) for domains, or global (cover whole protein)

PfamPfam


TIGRfamTIGRfam

Full length alignments. Domain alignments. Equivalogs: families of

proteins with specific function.

Superfamilies: families of homologous genes.

HMMs

http://www.tigr.org/TIGRFAMs/


KEGG orthologyKEGG orthology


Composite pattern Composite pattern databasesdatabases

To simplify sequence analysis, the family databases are being integrated to create a unified annotation resource – InterPro

Release 30.0 (Dec10) contains 21178 entries Central annotation resource, with pointers to its satellite dbs

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


* It is up to the user to decide if the annotation is correct *


ENZYMEENZYME


http://ca.expasy.org/enzyme/

ENZYMEENZYME


KEGGKEGG

Contains information about biochemical pathways, and protein interactions.

http://www.kegg.com


Functional annotationFunctional annotation

COGsCOGs

genegene

KO termsKO terms

PfamTIGRfam

PfamTIGRfam

IMGIMG

PSI BLAST1e-2

BLASTp<1e-10, >45% id,

>70% length

Hmmsearch(BLAST preprocessing)

BLASTpevalue<10, 20 best hits

IMG termIMG term

TIGRfamTIGRfam

COGCOG

COG + pfamCOG + pfam

PfamPfam

Product name (based on translation tables)

NO

NO

NO

NO

YES

YES

YES

YES

YES

hypothetical

NO

BLASTBLAST

NO

YES

http://imgweb.jgi-psf.org/img_er_v260/doc/img_er_ann.pdf


Sequencing projects Sequencing projects GOLD

Information for ongoing and finished (meta)genomic projects.

Information about the metadata of genomes and metagenomic samples.

http://www.genomesonline.org


Literature searchLiterature search

PubMed

http://www.ncbi.nlm.nih.gov/Pubmed


Specialized Specialized databasesdatabases

There is a large number of databases devoted to specific organisms.

For some model organisms there are often concurrent systems.

These databases are associated to sequencing or mapping projects.


Other specialized databasesOther specialized databases

Signal transduction, regulation, protein-protein interactions TRANSFAC (Transcription Factor database) BRITE (Biomolecular Relations in Information Transmission and

Expression database) DIP (Database of Interacting Proteins) BIND (Biomolecular Interaction Network database) BioCarta

Biochemical pathways KLOTHO (Biochemical Compounds Declarative database) BRENDA (enzyme information system) LIGAND (similar to Enzyme but with more information for

substrates) Gene order and co-occurrence

STRING

3D structuresPDB (Protein Data Bank)MMDB (Molecular Modelling Data Base)NRL_3D (Non-Redundant Library of 3D Structures)SCOP (Structural Classification of Proteins)

PolymorphismALFRED (Allele Frequency Database)

Molecular interactionsDIP (Database of Interacting proteins)BIND (Biomolecular Interaction Network Database)

Gene expressionGXD (Mouse Gene Expression Database)The Stanford Microarray Database

MappingGDB (Genome Data Base)EMG (Encyclopedia of Mouse Genome)MGD (Mouse Genome Database)INE (Integrated Rice Genome Explorer)

Protein quantificationSWISS-2DPAGEPDD (Protein Disease Database)Sub2D (B. subtilis 2D Protein Index)


List of databasesList of databases

http://www.oxfordjournals.org/nar/database/c


Databanks Databanks interconnectioninterconnection

SWISS-PROT

ENZYME

PDB

HSSP

SWISSNEW

YPDREF

YPD

PDBFINDERALI

DSSP

FSSP

NRL_3D

PMD

PIR

ProtFam

FlyGene

TFSITE

TFACTOR

EMBL

TrEMBL

ECDC

TrEMBLNEW

EMNEW

EPD

GenBank MOLPROBE

OMIM

MIMMAP

REBASE

PROSITE ProDom

PROSITEDOCBlocks

SWISSDOM

Not all databases are updated regularly. Changes of annotation in one database are not reflected in others.


Summary Summary

We have main archives (Genbank), and currated databases (Refseq, SwissProt), and protein classification database (COG, Pfam), and many, many more…

They help predict the function, or the network of functions.

Systems that integrate the information from several databases, visualize and allow handling of data in an intuitive way are required


Thank you for your attention.

Documents

Functional annotation Datasources Konstantinos Mavrommatis Kmavrommatis@lbl