A deep audit of the PeptideAtlas database uncovers evidence for unannotated coding genes and aberrant translation

The human genome has been the subject of intense scrutiny by experimental and manual curation projects for more than two decades. Novel coding genes have been proposed from large-scale RNASeq, ribosome profiling and proteomics experiments. Here we carry out an in-depth analysis of an entire proteomics database. We analysed the proteins, peptides and spectra housed in the human build of the PeptideAtlas proteomics database to identify coding regions that are not yet annotated in the GENCODE reference gene set. We find support for hundreds of missing alternative protein isoforms and unannotated upstream translations, and evidence of cross-contamination from other species. There was reliable peptide evidence for 34 novel unannotated open reading frames (ORFs) in PeptideAtlas. We find that almost half belong to coding genes that are missing from GENCODE and other reference sets. Most of the remaining ORFs were not conserved beyond human, however, and their peptide confirmation was restricted to cancer cell lines. We show that this is strong evidence for aberrant translation, raising important questions about the extent of aberrant translation and how these ORFs should be annotated in reference genomes.