Covering all your bases: incorporating intron signal from RNA-seq data

Abstract

RNA-seq datasets can contain millions of intron reads per library that are typically removed from downstream analysis. Only reads overlapping annotated exons are considered to be informative since mature mRNA is assumed to be the major component sequenced, especially for poly(A) RNA libraries. In this study, we show that intron reads are informative, and through exploratory data analysis of read coverage that intron signal is representative of both pre-mRNAs and intron retention. We demonstrate how intron reads can be utilized in differential expression analysis using our index method where a unique set of differentially expressed genes can be detected using intron counts. In exploring read coverage, we also developed the superintronic software that quickly and robustly calculates user-defined summary statistics for exonic and intronic regions. Across multiple datasets, superintronic enabled us to identify several genes with distinctly retained introns that had similar coverage levels to that of neighbouring exons. The work and ideas presented in this paper is the first of its kind to consider multiple biological sources for intron reads through exploratory data analysis, minimizing bias in discovery and interpretation of results. Our findings open up possibilities for further methods development for intron reads and RNA-seq data in general.

Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing

“…Since D10 is present in the nuclear fraction, we investigated whether pre-mRNAs might be targeted by D10 before intron removal as a mechanism to distinguish transcripts derived from intron-containing and intronless genes. Reads mapping to introns are an accurate readout of pre-mRNA levels [ 29 ]. While exonic reads were reduced upon WT D10 expression, intronic reads were unchanged ( Fig 3E ), suggesting that D10 does not target pre-mRNAs in the nucleus.…”

Genomic variants from RNA-seq for goats resistant or susceptible to gastrointestinal nematode infection

“…Our study revealed many variants in intronic regions. Indeed, it has been shown that the percentage of introns reads was higher when libraries were prepared with total RNA extraction methods, as in the present study, compared with the poly(A) RNA ones [ 40 ]. It should also be noted that the results presented here for variants position do not necessarily reflect the percentages of intron/exon, since variant effect prediction had not been made for all variants but only for those which differ between resistant and susceptible animals.…”

Cellular and genetic drivers of RNA editing variation in the human brain

“…Specifically, calling RNA editing sites from snRNA-seq is challenging as the technique is limited from extremely low capture efficiency and low sequencing depth with reads covering only a fraction of the entire genome 36,37 . Moreover, intronic regions, which were highly edited in FANS-derived data, often represent high vulnerability to low-coverage in scRNA-seq experiments, where detection of A-to-I events is reduced 38 . Validation rates by snRNA-seq were highest for GLU and MGE-GABA populations, which constituted pools with the largest number of nuclei, and concordance of editing levels for these sites was exceptionally high between snRNA-seq and FANS-derived data sets.…”