The Consensus CoDing Sequence (CCDS) Database Kim D. Pruitt Mouse Genome Annotation Summit Meeting March 12-13, 2008

Why is the CCDS project needed? The Problem: Annotation of the genome sequence is essential – but beware of different interpretations! The availability of the human and mouse genome sequence has had a significant impact on disease and health research. Most scientists rely on annotation information when designing, interpreting, and evaluating research results. Inconsistencies in annotation results among the main public resources hampers use of this important data. Researchers may not realize that a different annotation result is available elsewhere – possibly leading to erroneous or incomplete interpretations.

CCDS - A collaborative project Initiated by the main public annotation/browser groups to address concerns by the scientific community about inconsistencies in the human and mouse genome annotation. Built by consensus among the collaborating members, which include: European Bioinformatics Institute (EBI) National Center for Biotechnology Information (NCBI) University of California, Santa Cruz (UCSC) Sanger Institute (WTSI)

What is the CCDS project? Project Goals identify a core set of protein-coding genes that are consistently annotated and of high quality support convergence toward a standard set of gene annotations Scope: Human and mouse protein coding regions Update frequency Variable Depends on frequency of genome annotation updates

Process flow – calculating updates NCBI (computational) Havana (manual) Ensembl (computational) RefSeq (manual) Compare CDS (Annotation + Sequence) Ensembl merged annotation QA Identical Similar Novel Existing CCDS Retain Lost New match New CCDS ID Out of scope

Quality assessment tests include: Assessing Quality CCDS status is conservatively applied: Annotated CDS coordinates are identical Annotation is of high quality and passes QA tests, or curator review Existing CCDS proteins can be flagged for review by the collaborating members Updates and removals are by consensus agreement. Quality assessment tests include: Consensus splice sites ("GY..AG" or "AT..AC") Valid start and stop codons with no internal stops NMD Low complexity Repeat-containing Insufficient protein homology Genome conservation Putative pseudogene QA test results are reviewed by curators Over-rides are set to retain supported CDSs

CCDS Counts Date Build CDS IDs GeneIDs Mar-05 Hs35.1 14,795 13,142 Feb-07 Hs36.2 18,290 16,008 Oct-06 Mm36.1 13,374 13,014 Nov-07 Mm37.1 17,707 16,893 Step Source Genes Proteins Annotation NCBI 24765 26851 Annotation Ensembl 27209 39941 Matching CDS 18185 19048 QA & curation rejections 1331 1350 Accepted rejections 1292 1341 Final CCDS ID 16893 17707

Curation – how are updates curated and coordinated? Any member of the collaboration can flag a CCDS for review Update the CDS definition (alter N-terminus extent internal splice site) Withdraw the CCDS ID (insufficiently supported, or non-protein coding) NCBI provides a collaboration web site to coordinate this review All collaborators must agree with a change to finalize a decision Withdrawal of a CCDS may happen between genome annotation updates An update to a CCDS is indicated by: Status change: a status of ‘pending update’ is reported when there is collaborative agreement that a change is needed Version change: The CCDS version number is incremented once the change is reflected in public annotation. This only occurs after a genome annotation update and CCDS analysis has taken place. CCDS curation is fully integrated with RefSeq curation

CCDS update & curation stats Curation-based changes: Mouse: ~5200 curated CCDS genes name action status count human update pending 366 human update agreed 557 human withdraw pending 189 human withdraw agreed 519 mouse update pending 185 mouse update agreed 57 mouse withdraw pending 16 mouse withdraw agreed 8 923 709 242 24 Annotation pipeline-based changes: name build status count human 35.1 Withdrawn, inconsistent annotation 133 human 36.2 Withdrawn, inconsistent annotation 29 mouse 36.1 Withdrawn, inconsistent annotation 29 mouse 37.1 Withdrawn, inconsistent annotation 4

Curation considerations Alignments Track low quality sequences (‘kill list’) Protein conservation Publications Personal communications QA measures

Access – How do I know if an annotation has a CCDS ID? Genome browser displays NCBI UCSC Gene reports Ensembl Vega Other: RefSeq annotation (NCBI) CCDS web site FTP http://www.ncbi.nlm.nih.gov/CCDS/

NCBI Map Viewer (chr.5) Link to CCDS Browser

UCSC Browser chr5:30270000-30650000

UCSC Browser – Tyms gene CCDS Browser

Access of CCDS data at NCBI CCDS Database & Browser interface Project Description Query support Reports attributes of the CCDS Location data Sequence members Status FTP reports

CCDS Browser History Find all CCDSs for the Gene Entrez Gene View CCDS Details

CCDS Browser Mouse-over highlights codon Click to highlight codon and corresponding amino acid

Biology is complex – some CCDS curation examples 1 vs 2 vs ‘n’ genes translation start site

1 vs. 2 vs. ‘n’ genes Curation Considerations: Nomenclature History (scientific use, publications, etc.) Different (but similar) products vs. distinct products Shared promoters

carnitine palmitoyltransferase 1b, choline kinase beta

Current RefSeq representation of the region - two protein coding loci 1 vs. 2 vs. ‘n’ genes Current RefSeq representation of the region - two protein coding loci - one non-coding loci for the non-coding transcript product (a read-through transcript) Chkb (CCDS27750.1) Cpt1b (CCDS27749.1 ) Chkb-cpt1b (PMID:12761301 )

Translation start site Curation Considerations Publication reports (CDS begins at ‘n’) Other cDNA sequencing reveals the ORF can be extended further upstream Evaluate: Genome conservation Literature reports for the protein Putative Kozak signals Presence of in-frame upstream stop codon INSDC submissions from an experimental lab source that do have the longer ORF extent annotated. Consult with an expert

Internal CCDS browser (restricted access) Jmjd2d jumonji domain containing 2D (chr 19)

Update is agreed on by all parties Resulting in a 258 aa N-terminal extension

Examples – no CCDS ID EBI+WTSI and NCBI transcript annotation may differ even though the gene includes annotations with CCDS IDs

Examples –no CCDS ID Reasons: not found by one group different CDS length different splice sites different internal exon Curation removal EBI/WTSI NCBI EBI/WTSI NCBI EBI/WTSI NCBI EBI/WTSI NCBI

Acknowledgements Collaborators at Ensembl, UCSC, Vega Donna Maglott Josh Cherry Keith Oxenride Craig Wallin Andrei Shkeda RefSeq Curators NCBI Genome Annotation Group NCBI Map Viewer Group Collaborators at Ensembl, UCSC, Vega Jen Ashurst & Vega curator group Rachel Harte Mark Diekhans Steve Searle

Ensembl – Tyms gene

Vega browser Tyms gene (chromosome 5 30388989-30404404)