YeATSAM analysis of the chloroplast genome of walnut reveals several putative un-annotated genes and mis-annotation of the trans-spliced rps12 gene in other organisms

An open reading frame (ORF) is genomic sequence that can be translated into amino acids, and does not contain any stop codon. Previously, YeATSAM analyzed ORFs from the RNA-seq derived transcriptome of walnut, and revealed several genes that were not annotated by widely-used methods. Here, a similar ORF-based method is applied to the chloroplast genome from walnut (Accid:KT963008). This revealed, in addition to the ~84 protein coding genes, ~100 additional putative protein coding genes with homology to RefSeq proteins. Some of these genes have corresponding transcripts in the previously derived transcriptome from twenty different tissues, establishing these as bona fide genes. Other genes have introns, and need to be manually annotated. Importantly, this analysis revealed the mis-annotation of the rps12 gene in several organisms which have used an automated annotation flow. This gene has three exons - exon1 is ~28kbp away from exon2 and exon3 - and is assembled by trans-splicing. Automated annotation tools are more likely to select an ORF closer to exon2 to complete a possible protein, and are unlikely to properly annotate trans-spliced genes. A database of trans-spliced genes would greatly benefit annotations. Thus, the current work continues previous work establishing the proper identification of ORFs as a simple and important step in many applications, and the requirement of validation of annotations.