Widespread Occurrence of Hybrid Internal-terminal Exons in Human Transcriptomes

Messenger RNA isoform differences are predominantly driven by alternative first, internal, and last exons. Despite the importance of classifying exons to understand isoform structure, few tools examine isoform-specific exon usage. We recently observed that alternative transcription start sites often arise near internal exons, often creating “hybrid” first/internal exons. To systematically detect hybrid exons, we built the hybrid-internal-terminal (HIT) pipeline to classify exons depending on their isoform-specific usage. On the basis of splice junction reads in RNA sequencing data and probabilistic modeling, the HIT index identified thousands of previously misclassified hybrid first-internal and internal-last exons. Hybrid exons are enriched in long genes and genes involved in RNA splicing and have longer flanking introns and strong splice sites. Their usage varies considerably across human tissues. By developing the first method to classify exons according to isoform contexts, our findings document the occurrence of hybrid exons, a common quirk of the human transcriptome.