Human splicing diversity and the extent of unannotated splice junctions across human RNA-seq samples on the Sequence Read Archive

Abstract

BackgroundGene annotations, such as those in GENCODE, are derived primarily from alignments of spliced cDNA sequences and protein sequences. The impact of RNA-seq data on annotation has been confined to major projects like ENCODE and Illumina Body Map 2.0.ResultsWe aligned 21,504 Illumina-sequenced human RNA-seq samples from the Sequence Read Archive (SRA) to the human genome and compared detected exon-exon junctions with junctions in several recent gene annotations. We found 56,861 junctions (18.6%) in at least 1000 samples that were not annotated, and their expression associated with tissue type. Junctions well expressed in individual samples tended to be annotated. Newer samples contributed few novel well-supported junctions, with the vast majority of detected junctions present in samples before 2013. We compiled junction data into a resource called intropolis available at http://intropolis.rail.bio. We used this resource to search for a recently validated isoform of the ALK gene and characterized the potential functional implications of unannotated junctions with publicly available TRAP-seq data.ConclusionsConsidering only the variation contained in annotation may suffice if an investigator is interested only in well-expressed transcript isoforms. However, genes that are not generally well expressed and nonetheless present in a small but significant number of samples in the SRA are likelier to be incompletely annotated. The rate at which evidence for novel junctions has been added to the SRA has tapered dramatically, even to the point of an asymptote. Now is perhaps an appropriate time to update incomplete annotations to include splicing present in the now-stable snapshot provided by the SRA.

The landscape of human mutually exclusive splicing

“…Extrapolating these observations to all splice types and genes suggests the existence of thousands yet unannotated exons in introns. This estimation is in accordance with a recent analysis of more than 20,000 human RNA‐Seq datasets that revealed over 55,000 junctions not present in annotations (Nellore et al , 2016). In this analysis, junctions found in at least 20 reads across all samples were termed “confidently called”.…”

Evolutionarily Conserved Alternative Splicing Across Monocots

“…These are treasure troves of data waiting to be mined for new discoveries. Many are already tapping into this wealth, as exemplified by Nellore et al (2016), who examined splice junction variants in the human genome, and Chamala et al (2015) who examined conserved AS events across several species of eudicots. The method we describe here can be used to mine to identify conserved AS events within any species with an available genome sequence and deep RNA-Seq data.…”

Whippet: an efficient method for the detection and quantification of alternative splicing reveals extensive transcriptomic complexity

“…Our results from applying Whippet indicate that high-entropy AS events occur more frequently in vertebrate transcriptomes than previously appreciated 12,31 , and further provide evidence that these events are likely biologically significant since their entropy levels are frequently tissue-regulated, conserved, and the corresponding variant transcripts are highly expressed. Many of the events are reminiscent of wellstudied examples of high-entropy AS in other systems, such as the myriad of splice variants generated by tandem arrays of alternative exons in the Drosophila DSCAM gene 32 .…”