Quality assessment of splice site annotation based on conservation across multiple species

Abstract

Despite many improvements over the years, the annotation of the human genome remains imperfect, and even the best annotations of the human reference genome sometimes contradict one another. Hence, refinement of the human genome annotation is an important challenge. The use of evolutionarily conserved sequences provides a strategy for addressing this problem, and the rapidly growing number of genomes from other species increases the power of an evolution-driven approach. Using the latest large-scale whole genome alignment data, we found that splice sites from protein-coding genes in the high-quality MANE annotation are consistently conserved across more than 400 species. We also studied splice sites from the RefSeq, GENCODE, and CHESS databases that are not present in MANE, from both protein-coding genes and lncRNAs. We trained a logistic regression classifier to distinguish between the conservation patterns exhibited by splice sites from MANE versus sites that were flanked by the standard GT-AG dinucleotides, but that were chosen randomly from a sequence not under selection. We found that up to 70% of splice sites from annotated protein-coding transcripts outside of MANE exhibit conservation patterns closer to random sequence as opposed to highly-conserved splice sites from MANE. Our study highlights potentially erroneous splice sites that might require further scrutiny.