Paired-end small RNA sequencing reveals a possible overestimation in isomiR sequence repertoire previously reported from conventional single read data analysis

Next generation sequencing has allowed the discovery of miRNA isoforms, termed isomiRs. Some isomiRs are derived from imprecise processing of pre-miRNA precursors, leading to length variants. Additional variability is introduced by non-templated addition of bases at the ends or editing of internal bases, resulting in base differences relative to the template DNA sequence. We hypothesized that some component of the isomiR variation reported so far could be due to systematic technical noise. We have developed the XICRA pipeline to analyze small RNA sequencing data at the miRNA,. We exploited its ability to use single or merged reads to compare miRNA isomiR results derived from paired-end (PE) reads with those from single reads (SR) to address whether detectable sequence differences relative to canonical miRNAs found in isomiRs are true biological variations or the result of errors in sequencing. We have detected non-negligible systematic differences between single and PE data which primarily affect putative internally edited isomiRs, and at a much smaller frequency terminal length changing isomiRs. This is relevant for the identification of true isomiRs in small RNA sequencing datasets. . We conclude that potential artifacts derived from sequencing errors and/or data processing could result in an overestimation of abundance and diversity of miRNA isoforms. Efforts in annotating the isomiRnome should take this into account. This is an mRNA sequencing study which addressed the effect of paired end sequencing versus single read sequencing on miRNA and isomir expression profiling by next generation sequencing. A total of 28 serum derived total RNA samples were proifiled in the study derived form fourteen male and fourteen of female individuals. The small RNA fraction was sequenced using Illumina technology generating reads from both ends of the library. Differential expression was assessed by comparing normalized sequencing read count levels between the two genders. We focused on the differences in the results derived from using single reads versus joined read pairs.