Genome Annotation and
Databases
Genomic DNA sequence
Genomic annotation
Reading Ch 9, Ch10
BIO520 Bioinformatics
Jim Lund
Genome Annotation
• Find known repeats
• Search for new repeated sequences
• Predict Genes
– BLASTX
– Genewise, Fgenes,
Genscan…
• Integrate other data
sources.
Accuracy highest in “high homology” class
Genome annotation servers
• Integrate information from several maps
– DNA sequence (contigs, quality).
– Physical (cytogenetic, STS content).
– Genes (show gene annotations and evidence).
• Several prediction programs.
• Expressed sequence tags (ESTs, Unigene
clusters)
• Evidence (Predicted, confirmed)
• Non-coding RNA (ncRNA) transcripts.
– Variation (e.g., SNPs)
– Regions of shared synteny.
Data Release
• Human genome sequence released under 1996
Bermuda rules
– Assembled sequence greater than 1000bp long is
deposited in public database (GenBank/EMBL/DDBJ)
every 24 hours
– No patents are filed
• Bermuda principles reaffirmed at January 2003
WT/NIH meeting
– Pre-release of data for all “community projects”
– Nature 421 , 875 (2003)
– NHGRI:
• http://www.genome.gov/page.cfm?pageID=10506376
– WT:
• http://www.wellcome.ac.uk/About-us/Policy/Policy-andposition-statements/WTD002751.htm
• Benefits of Open Data Access supported by
OECD report
– http://dataaccess.ucsd.edu
Accessing the Genome
• Genomes sequences are becoming available very rapidly
– Large and difficult to handle computationally
– Everyone expects to be able to access them immediately
• Bench Biologists
–
–
–
–
Has my gene been sequenced?
What are the genes in this region?
Where are all the GPCRs
Connect the genome to other resources.
• Research Bioinformatics
– Give me a dataset of human genomic DNA.
– Give me a protein dataset.
Getting information out
• Search/browse to find the gene or
region.
• Export formats:
– Screen shot
– FASTA seq.
– Genbank file with features annotated
– Feature list (Gff, tab-delimited text)
– Pip (plot of sequence identity between
organisms).
Challenges
• Scale and data flow
– Presentation, ease of use.
– Engineering problems.
– User interface design.
• Algorithmic
– Partly engineering (pre-compute hard
computations, etc.)
– Partly research.
NCBI sequence assembly
(sequence
chromosome)
• Remove contaminants
• Bin by chromosome arms
• Sequence Layout
• Sequence Building
• Place on chromosomes
http://www.ncbi.nlm.nih.gov/genome/guide/build.shtml
NCBI sequence assembly - a modified greedy
approach
Sequence Layout
•Curated Finished Regions
•Curated assembly instructions
•MegaBLAST hits
•Consider clone order
•BAC chromosome assignment
•annotation
•STS markers
•personal communication
•Remove conflicting overlaps, redundant BACs
Sequence Building
•Consider fragment:fragment sequence overlaps for each BAC pair in
layout
•Meld overlapping sequence
•Order and Orient (o+o ):
•alignments (mRNA, EST)
•BAC annotation
•paired plasmid reads
BAC Sequence
Fragments
Assemble
Order
NCBI Contig
NCBI Genome Build Process
dbSNP
STS
Clones
GenomeScan
Collaboration
Curation
GenBank
LocusLink
RefSeq
LocusLink
Assembly
Contig Build
&
Release
Freeze
Input Data:
Sequences
Curated NTs
TPF
BLAST hits
Exclude
Problem
accessions
Annotation
Update:
Links
gi’s
Prepare for release
Resource
Updates
Public Release
Analysis & Review
Corrections for
next build
Sequences (contig mRNA protein)
Map Viewer
FTP
BLAST
Input Resources
What is being annotated?
Feature
Genes:
Method
By alignment, by prediction
Markers:
By ePCR
Variation:
By alignment
Clones/Cytogenetic location:
Phenotype (MIM):
Cytogenetic Position:
By alignment (BAC ends)
Via Gene identification, associated markers
By annotated BAC-END sequenced clones
By FISH-mapped clones used in assembly
RefSeq: a reagent for Contig Annotation
genome
RefSeq mRNAs
GenBank mRNAs
ESTs
TBLASTN
RPSBLAST
GenomeScan
Potential Problems
With ESTs:
•Gene Families
•Partial
•Chimeric
•Intron read-through
•Linker
•Vector
•Wrong organism
RefSeq Advantages:
•Separate Gene Families
•Not Partial
•Means to correct
problem sequences
RefSeq process results
in excluding problem
GenBank sequences
from annotation pipeline
NCBI: Products of annotation
•
•
•
•
RefSeqs (transcripts, proteins)
Gene id (LocusID)
features in chromosome coordinates
features in contig (NT accession)
coordinates
Available in:
• Map Viewer
– Graphical display
– Tabular display
– Sequence downloads
• FTP
– RefSeqs (contigs, transcripts, proteins)
– Mapping Data
– LocusLink & Other resources
NCBI Map Viewer
NCBI Map Viewer: Tabular report
Genes in regions of conserved synteny
Anchored by
human gene
order
Anchored by
mouse gene order
Chromosomal segments in dog
conserved with human and mouse
Dog: 38 autosomes + sex chr
Query by sequence: Review the alignment
A click away:
•Alignments (BLAST hit)
•Gene Description
(LocusLink)
•Report of all features
in the region
•Contig sequence
•Sequence in the region
•other mRNAs aligning in
the region
•Define your own gene
model based on
alignments in the region
Quality Control - Genome review
•
•
•
•
Is the sequence correct?
Is the feature correctly placed?
Is there a feature that should be placed?
Are the attributes of the feature correct?
Approaches:
•In-house analysis & review (manual curation)
•Shared information (UCSC/Ensembl)
•Solicited review by experts in local regions
Ensembl Annotation pipeline
• Set of high quality gene predictions
– From known human mRNAs aligned against genome
– From similar protein and mRNAs aligned against genome
– From Genscan predictions confirmed via BLAST of Protein,
cDNA, ESTs databases.
• Initial functional annotation from Interpro
• Integration with external resources (SNPs, SAGE,
OMIM)
• Comparative analysis between mouse/human
– DNA sequence alignment
– Protein orthologs
Ensembl gene prediction pipeline
DNA
RepeatMasker
Genscan
Blast genscan peptides v
Protein,unigene,est,vert mrna
MiniGenewise
MiniEst2genome
Genes
Pmatch all human
Proteins and cdnas
Genome Annotation
The generic structure of an automatic genome annotation pipeline and delivery system
Chromosome
Overview
Genes and Markers
1Mb
Configuration
Detailed View
Genes, ESTs, CpG etc.
100kb
Useful genomic annotation and
browser URLs
EBI/Sanger Institute Ensembl Project:
http://www.ensembl.org/Homo_sapiens/
NCBI Human Genome Browser:
http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?chr=hum_chr.inf&query
The Oak Ridge National Laboratories Genome Channel:
http://compbio.ornl.gov/channel/
UCSC Human Genome Browser:
http://genome.ucsc.edu/cgi-bin/hgGateway
The Institute for Genomic Research (TIGR):
http://www.tigr.org/
Genome annotation
-things still being worked out-
•Annotation servers.
•Pro: make genomics information accessible to biologists without expert
bioinformatics skills.
•Con: makes it difficult to perform large-scale data mining.
•Solution: enable more experienced users to retrieve the data they require
and to run analyses locally.
•Open annotation systems.
•Biologists need to have access to annotations available in the community
and to share their own contributions with the community.
•A common protocol between systems that enables genome data to be
freely exchanged
•AGAVE (Architecture for Genomic Annotation, Visualization and
Exchange)
•Distributed Annotation System (DAS) projects
Genome annotation servers
• Several ways to find information:
– Search by clone, gene, EST, marker.
– Browse sequence.
– BLAST searches.
– Homology, start in one organism, jump
to the syntenic region of another.
UCSC Genome Browser
http://genome.ucsc.edu/cgi-bin/hgGateway