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Evaluating Genes and TranscriptsEvaluating Genes and Transcripts(“Genebuild”)(“Genebuild”)

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OutlineOutline

• Ensembl gene set

• Ensembl EST genes

• Ab initio predictions

• Manual curation (Vega)

• Ensembl / Havana merged gene set

• CCDS project

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Biological EvidenceBiological Evidence

• UniProt/Swiss-ProtA manually curated database and therefore of highest accuracy

• NCBI RefSeqA partially manually curated database

• UniProt/TrEMBLAutomatically annotated translations of EMBL coding sequence (CDS) features

• EMBL / GenBank / DDBJPrimary nucleotide sequence repository

All Ensembl gene predictions are based on experimental evidence:

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The Ensembl GenebuildThe Ensembl Genebuild

Genome assembly

Computer programs

Experimental evidence

Ensembl Ensembl GenesGenes

+

+

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The Ensembl GenebuildThe Ensembl Genebuild

A new release of Ensembl doesn’t contain a new genebuild for each species!

New genebuilds are only done if there is:• a new genome assembly• a lot of new supporting evidence

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Genome AssembliesGenome Assemblies

Genome assemblies are not created byEnsembl, but provided by other institutes / consortia, e.g.

• NCBI: human, mouse• Rat Genome Sequencing Consortium: rat• Sanger: zebrafish• Broad Institute: mammals• Baylor College: cow• Washington University: chicken

etc. etc.

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The Ensembl GenebuildThe Ensembl Genebuild

• Targeted build:

Align species-specific proteins to the genome to create transcripts

• Similarity build:

Align proteins from closely related species to locate additional transcripts

• Add UTRs using mRNA evidence• Eliminate redundant transcripts and create

genes

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““Special” casesSpecial” cases

• Pseudogenes

• Non-coding RNA genes

sequences from RFAM and miRBase dbs and covariance modelshand-checked set

• Ig Segment Genes (Immunoglobulin and T-cell receptor segments)

sequences from IMGT db and Exonerate

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Classification of TranscriptsClassification of Transcripts

• Ensembl Transcripts and Proteins are mapped to UniProt/Swiss-Prot, NCBI RefSeq and UniProt/TrEMBL entries

• Genes that map to species-specific protein/mRNA records are classified as known

• Genes that do not map to species-specific protein/mRNA records are classified as novel

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Names and DescriptionsNames and Descriptions

• Transcript names are inferred from mapped transcripts and proteins• Swiss-Prot > RefSeq > TrEMBL ID

• Novel transcripts have only Ensembl identifiers

• Genes are assigned the official gene symbol if available• HGNC (HUGO) symbol for human genes• Species-specific nomenclature committees (MGI,

ZFIN etc.)

• Otherwise Swiss-Prot > RefSeq > TrEMBL ID• Gene description is inferred from mapped

database entries, the source is always given

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Supporting evidenceSupporting evidence

ExonView

mRNA

peptidepeptide

mRNA

UTR coding/UTR

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Supporting evidenceSupporting evidenceContigView

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Configuring the GenebuildConfiguring the Genebuild

Genebuild configured for each species

Data availibility• Targeted build most useful in human, mouse• Similarity build most useful in C. intestinalis,

mosquito

Structural issues• Zebrafish

• Many duplications• Genome from different haplotypes

• Mosquito• Many single-exon genes• Genes within genes

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Low Coverage GenomesLow Coverage Genomes

• Low coverage genomes (~2x) come in lots of scaffolds: “classic” genebuild will result in many partial and fragmented genes

• Whole Genome Alignment (WGA) to an annotated reference genome: this method reduces fragmentation by piecing together scaffolds into “gene-scaffolds” that contain complete gene(s)

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Low Coverage GenomesLow Coverage Genomes

NNNNNN “gene-scaffold”

reference assembly

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EST Gene SetEST Gene Set

• ESTs (Expressed Sequence Tags) are single reads, high chance of sequencing mistakes

• EST libraries are regularly contaminated with genomic DNA

• Generally ~ 400 bp, so unlikely to cover a whole gene

THEREFORE

• EST gene predictions are less reliable and thus kept separate from the core Ensembl Gene Set

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EST Gene SetEST Gene SetContigView

ESTs

EST genes

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Ab initioAb initio Predictions Predictions

• Predict translatable transcript structures solely on the basis of genome sequence.

• No validation with biological expression information.

• GENSCAN for vertebrate genomes• SNAP better for invertebrates• NB: Both programs are over-

predicting transcript structures.

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Ab initioAb initio Predictions PredictionsContigView

GENSCAN prediction

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Automatic vs Manual Automatic vs Manual AnnotationAnnotation

Automatic Annotation• Quick• Use unfinished

sequence or shotgun assembly

• Consistent annotation

Manual Annotation• Slow• Need finished

sequence• Flexible, can deal

with inconsistencies• Most rules have

exceptions• Consult publications

as well as databases

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Annotation that Causes Problems Annotation that Causes Problems for Ensemblfor Ensembl

• Multiple variants

• UTRs

• Pseudogenes

• Non-coding genes (ncRNAs)

• Overlapping genes, anti-sense genes

• Gene duplication events

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Manually Curated Gene SetsManually Curated Gene Sets

• FlyBase fruitfly

• WormBase C. elegans

• SGD yeast

• Vega human, zebrafish, mouse, dog

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Vega Genome BrowserVega Genome Browser

http://vega.sanger.ac.uk

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Vega TranscriptsVega Transcripts

Vega transcripts

Vega Havana transcripts annotated by the Havana team at SangerVega External transcripts annotated by other Vega teams

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Ensembl / Havana MergeEnsembl / Havana Merge

Transcripts:• Ensembl/Havana: gold • Ensembl: red / black• Havana: blue

Genes: • Ensembl/Havana: gold • Ensembl: red / black• Havana: blue

Full-length protein-coding transcripts annotated by the Sanger Havana team (part of Vega) are merged with the human Ensembl transcript set

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Ensembl / Havana MergeEnsembl / Havana Merge

Merged Ensembl / Havana gene

Merged Ensembl / Havana transcript

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CCDSCCDS((CConsensus onsensus CCoodding ing SSequences)equences)

• Collaboration between NCBI, UCSC, Ensembl and Havana to produce a set of stable, reliable, complete (ATG->stop) CDS structures for human and mouse

• Long term aim is to get to a single gene set for human and mouse

• The genebuild pipeline has been modified to retain these ‘blessed’ CDSs (stored in a database for incorporation in the build)

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