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. Overall design: 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.

下一代测序技术推动了miRNA异构体（isomiRs）的发现。部分isomiRs源于pre-miRNA前体的不精确加工，进而产生长度变异体；另有部分变异源自末端非模板化碱基添加或内部碱基编辑，导致其碱基序列与模板DNA序列存在差异。我们推测，目前已报道的部分isomiR变异可能源于系统性技术噪声。

我们开发了XICRA分析流程，用于在miRNA层面开展小RNA测序数据分析。我们利用该流程可使用单端测序（SR）读段或合并读段的特性，对比由双端测序（PE）读段与单端测序（SR）读段得到的miRNA isomiRs分析结果，以探究isomiRs中相较于经典miRNA的可检测序列差异，究竟是真实的生物学变异，还是测序错误所致。

我们发现单端与双端测序数据间存在不容忽视的系统性差异，这类差异主要影响推测的内部编辑型isomiRs，而对末端长度变异型isomiRs的影响频率则低得多。这一发现对小RNA测序数据集中真实isomiRs的鉴定具有重要参考价值。

我们由此得出结论：测序错误和/或数据分析流程所引入的潜在测序伪影，可能会导致miRNA异构体的表达丰度与多样性被高估。在注释isomiR组（isomiRnome）的相关工作中，应将这一因素纳入考量。

整体实验设计：本研究为mRNA测序研究，旨在通过下一代测序技术，探究双端测序与单端测序对miRNA及isomiR表达谱分析的影响。本研究共分析了28份血清来源的总RNA样本，这些样本来自14名男性与14名女性个体。研究采用Illumina测序技术对小RNA组分进行测序，获取文库两端的读段序列。通过比较两个性别群体间标准化后的测序读段计数水平，完成差异表达分析。本研究重点关注单端读段与合并读段对分析结果产生的差异。