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

Abstract

Background 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 and not real. Results We have developed the XICRA pipeline to analyze small RNA sequencing data at the isomiR level. We exploited its ability to use single or merged reads to compare 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 SR 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. Conclusions 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.

MicroRNA Isoforms Contribution to Melanoma Pathogenesis

“…In the literature, there is a concern that sequencing and/or mapping artefacts may result in an overrepresentation of isomiRs [30]. However, previous studies have shown that isomiRs represent actual molecules rather than sequencing artifacts.…”

Circulating Exosome Cargoes Contain Functionally Diverse Cancer Biomarkers: From Biogenesis and Function to Purification and Potential Translational Utility

“…Quantitative PCR technology is still considered the gold standard for validation of findings made during high-throughput analyses; however, it has inherent biological and experimental limitations for exosomal miRNA studies, which include: (1) The discovery of tumor miRNA panels specific to a tumor exosome remains limited to known or selected miRNAs (which can be multiplexed for up to 700 miRNAs using TaqMan card arrays) rather than global unbiased NGS analyses. In this fashion it does not allow discovery of IsomiR (i.e., miRNA variants of the same miRNA, which can differ in sequence and/or length composition and will bind to different mRNA targets than the original miRNA sequence [ 306 ]) expression ratio differences for a specific miRNA in exosome sub-populations. The study of isomiRs is relevant in cancer, as their expression ratio has been shown to be affected by and can elicit changes in tumor cell migration and invasion [ 307 ].…”

Emerging Role of isomiRs in Cancer: State of the Art and Recent Advances

“…NGS offers a great opportunity to specifically and sensitively detect isomiRs. However, the analysis of isomiRs remains a challenge, due to the limited number of specific tools available to analyze their expression levels in sequencing experiments and the lack of comparison among them [47] as well as the possible overestimation of isomiRs detected [99].…”