1. NCBI’s Genome Annotation: Overview Incremental processing Re-annotation ( batch ) Post-annotation review Case studies NOTE: limiting discussion to annotation of genes and pseudogenes Donna Maglott, Ph.D. for the RefSeq and Annotation groups

2. NCBI: Incremental processing Maintain gene/sequence relationship –Data sources MGI’s ftp site (names, MGI ids, sequence accessions) Sequences and annotation from INSD ( DDBJ, EMBL, GenBank ) UniProt (names, sequence) CCDS Collaboration (CDS definition; Kim will expand on this later.) Gene family-specific databases HomoloGene UniGene Scientific community –Actions Create or update data via Entrez Gene Create or update RefSeq sequences Identify conflicts and discuss with stakeholders

3. Create or update RefSeq sequences http:// www.ncbi.nlm.nih.gov/projects/sviewer /?id=NG_007044&v=947227:11232 Curated annotation of the T-cell receptor alpha/delta locus

4. Changed annotation after release of Mm37.1 Annotated as version 3, overlapping model at 5’ end

6. Hi MGI, I believe Tnrc18 (geneID: 381742, MGI:3648294) and Zfp469 (geneID: 231861, MGI:2448535) should be merged. This region of chr 5 has major assembly problems in the reference assembly, but the Celera assembly appears to accurately represent the structure as compared to transcript data and the orthologous regions of the human and rat genomes. In the reference assembly, Zfp469 and Tnrc18 are on separate scaffolds… there are multiple mouse and human transcripts spanning both loci… Zfp469 is currently represented as NM_178242.2 (based on BC049818.1), and this appears to be a valid transcript variant that uses a well-supported early polyA signal/site. However, mouse AK141849.1, CB249799.1, AK173280.1, and human AB058759.1 all extend past this early polyA signal/site to include an additional 13 exons that overlap with Tnrc18 and potentially encode an additional 1125 aa at the C-terminus…There is also an issue of nomenclature. I cannot find any evidence of the transcripts associated with Zfp469 encoding a zinc finger protein…the long variant does contain a trinucleotide repeat so the Tnrc18 name may be more appropriate, although the repeat is not found in the shorter NM_178242.2 variant. Also be aware that we had mis-associated the human TNRC18 nomenclature (HGNC: 11962).. -------- Terence Murphy Identify conflicts and discuss with stakeholders

7. One Gene or Two? BLAST alignment of human RefSeq NM_001080495.2 to Mm37.1

8. Exon coverage better in Celera assembly BLAST alignment of human RefSeq NM_001080495.2 to Celera

9. NCBI: Incremental processing Maintain gene/sequence relationship (cont’d) –Products RefSeq sequences via –Entrez Nucleotide, –Entrez Protein, –ftp Gene-specific data via –Entrez Gene –ftp Nomenclature propagated to UniGene and HomoloGene

10. NCBI: Re-annotation of genes Timing –Always with a new assembly –May occur without a re-assembly Evidence used –cDNAs aligned to the genome by Splign –Proteins aligned to the genome by proSplign –Ab initio predictions (gnomon) –Annotated RefSeq genomic sequences

11. NCBI: Re-annotation Tracking/identification of annotation ( decreasing weight ) –Best RefSeq placement (Splign/global alignment) –Comparison to previous annotation Assembly to assembly Clone to clone ‘product’ to ‘product’ –Best GenBank placement Products of annotation –If tracked, reassign GeneID and RefSeq model accession(s) –If novel and transcribed, assign new GeneID and RefSeq model accession(s) –If novel pseudogene, assign new GeneID and annotate on the genomic sequence without assigning a model RefSeq accession

12. One Gene or Two? BLAST alignment of human RefSeq NM_001080495.2 to Mm37.1

13. NCBI: Post annotation review Data reviewed –GeneIDs with sequence data, not annotated –GeneIDs annotated previously, not in the current annotation –CDS features NOT included in the CCDS set Actions taken –Create new RefSeqs if necessary –Update existing RefSeqs if necessary –Provide annotation comment to explain cases currently under review

14. NCBI: Post annotation review

15. NCBI: Representative review cases Under-representation of ncRNAs in the RefSeq set –May result in failure to annotate ncRNA that overlap a protein coding gene –More RefSeqs for this category are being generated Management of ‘read-through’ transcripts –Generate RefSeq if multiple lines of evidence –Discuss with all nomenclature groups

16. NCBI: Representative review cases Adjudication of the name to be assigned to a given genomic location –Evaluate conserved synteny –Discuss with all nomenclature groups

17. A case history: Arhgap27 and 5730442P18Rik VEGA: One Gene NCBI/MGI: Two Genes

18. A case history: Arhgap27 and 5730442P18Rik Contributing factors Computation: model prediction suggests one gene, but independent RefSeq mRNAs force two Only one gene is in the CCDS set Curation: NCBI merged in 2005 Reversed the merge in 2006 in response to request from MGI UniProt treats as one gene, Arhgap27 Evidence: one cDNA in rat, one cDNA from mouse, Arhgap12 all consistent with the one-gene model

19. A case history: A read-through locus http://www.ncbi.nlm.nih.gov/projects/sviewer/?id=NG_006051&v=2242:4418;g&p=theme:0&m=1

20. A case history: A (vertical) read-through locus http://tinyurl.com/2oy36j

21. A case history: olfactory receptors Action: Merge the Gene records in collaboration with MGI

22. A case history: interspersed loci http://www.ncbi.nlm.nih.gov/projects/sviewer/?id=NC_000073.5&v=52381146-52384105