Small RNA-Seq analysis of miRNA isoform (isomiR) expression in single porcine Day 10 blastocysts and analysis of effects of maternal estradiol-17-beta feeding from day of ovulation until Day 10 of pregnancy

Small RNA sequencing of 36 porcine embryo samples collected on Day 10 of pregnancy detected miRNA sequences mapping to known and predicted porcine miRNAs, and novel miRNAs highly conserved in human and cattle. A set of highly abundant miRNAs was identified as well as a large number of rarely expressed miRNAs. Due to the high number of generated sequence reads, it was possible to detect a large number of different miRNA isoforms (isomiRs). Potentially embryo-specific miRNAs were identified by comparing the data with small RNA-Seq data sets from porcine endometrium and a search in databases for miRNA tissue expression. The identification of 140 novel miRNAs in addition to 173 known porcine miRNAs (miRBase21) showed the benefit of our small RNA analysis pipeline. This benefit has a big impact for poorly annotated species such as the pig where it is possible to annotate many additional miRNAs based on the knowledge from related species. Furthermore, we integrated our pipeline into a Galaxy workflow using standard Galaxy tools which guaranties high reproducibility and flexibility for new/upcoming datasets/studies. Using ortholog species information increases the total number of annotated miRNAs while mapping to other non-coding RNAs decreased the chances of falsely annotated miRNAs. Galaxy workflows can be published, shared, downloaded, and optimized to adapt to other species or modify analysis parameters, such as tools specifically developed for a given research project/problem, so that a broad audience can use it.

本研究对妊娠第10天采集的36份猪胚胎样本开展小RNA测序（small RNA sequencing），成功检测到可比对至已知及预测猪源微小RNA（microRNA, miRNA）的序列，以及在人类和牛中高度保守的新型miRNA。研究同时鉴定出一组高丰度miRNA，以及大量低表达miRNA。由于本次测序产生的读段数量庞大，得以检测到大量不同的miRNA异构体（isomiRs）。通过将本研究数据与猪子宫内膜的小RNA测序（small RNA-Seq）数据集进行比对，并检索miRNA组织表达数据库，研究人员鉴定出潜在的胚胎特异性miRNA。本次研究共鉴定出173个已知猪源miRNA（源自miRBase数据库第21版，miRBase21），并新增140个新型miRNA，证实了本研究开发的小RNA分析流程的应用价值。这一优势对于注释信息匮乏的物种（如猪）具有重要意义——借助近缘物种的研究数据，可对大量额外miRNA完成注释。此外，本研究将该分析流程整合至基于标准Galaxy工具搭建的Galaxy工作流（Galaxy workflow）中，可为后续新增数据集或研究提供高度可复现性与灵活性。利用同源物种信息可提升注释得到的miRNA总数量，而通过将测序读段比对至其他非编码RNA（non-coding RNA）并筛选排除，可降低miRNA假注释的概率。Galaxy工作流可发布、共享、下载并优化，以适配其他物种或修改分析参数（例如为特定研究项目或问题开发的专用工具），从而可供广泛科研受众使用。