Mapping the RNA-Seq trash bin: unusual transcripts in prokaryotic transcriptome sequencing data

Abstract

Prokaryotic transcripts constitute almost always uninterrupted intervals when mapped back to the genome. Split reads, i.e., RNA-seq reads consisting of parts that only map to discontiguous loci, are thus disregarded in most analysis pipelines. There are, however, some well-known exceptions, in particular, tRNA splicing and circularized small RNAs in Archaea as well as self-splicing introns. Here, we reanalyze a series of published RNA-seq data sets, screening them specifically for non-contiguously mapping reads. We recover most of the known cases together with several novel archaeal ncRNAs associated with circularized products. In Eubacteria, only a handful of interesting candidates were obtained beyond a few previously described group I and group II introns. Most of the atypically mapping reads do not appear to correspond to well-defined, specifically processed products. Whether this diffuse background is, at least in part, an incidental by-product of prokaryotic RNA processing or whether it consists entirely of technical artifacts of reverse transcription or amplification remains unknown.

Keywords: RNA-seq; circular sRNAs; self-splicing introns; split tRNAs.

Figure 1. Density of circularized (thick line) and “spliced” reads (thin line) at the ribosomal rRNA loci. Coordinates refer to a multiple sequence alignment of the four Archaea species. For Nanoarchaeum, two separate RNA genes are concatenated with 160 Ns as linker.

Figure 2. Mapping of “introns” observed in multiple reads from E. coli (colored sequence), H. pylori (•) and P. aeruginosa (*) to the tmRNA structure (E. coli tmRNA model from ref. 31) shows that the excisions are concentrated in the pseudoknotted regions. Counts in brackets indicate the number of split reads.

Figure 3. Unusual eubacterial reads associated with tRNAs. Above: Spliced fusion of two adjacent tRNAs in H. pylori. Redmarks indicate the 5′-side of the acceptor stem and D-stem, resp. The apparent intron extends roughly from the end of the acceptor stem of tRNA-Ile to the beginning of the D-stem of tRNA-Ala. The coverage suggests that the two adjacent tRNAs are produced from a single primary transcript. Below: A circularized tRNA in Salmonella.