Despite the broad variety of available microRNA (miRNA) prediction tools, their application to the discovery and annotation of novel miRNA genes in domestic species is still limited. In this study we designed a comprehensive pipeline (eMIRNA) for miRNA identification in the yet poorly annotated porcine genome and demonstrated the usefulness of implementing a motif search positional refinement strategy for the accurate determination of precursor miRNA boundaries. The small RNA fraction from gluteus medius skeletal muscle of 48 Duroc gilts was sequenced and used for the prediction of novel miRNA loci. Additionally, we selected the human miRNA annotation for a homology-based search of porcine miRNAs with orthologous genes in the human genome. A total of 20 novel expressed miRNAs were identified in the porcine muscle transcriptome and 27 additional novel porcine miRNAs were also detected by homology-based search using the human miRNA annotation. The existence of three selected novel miRNAs (ssc-miR-483, ssc-miR484 and ssc-miR-200a) was further confirmed by reverse transcription quantitative real-time PCR analyses in the muscle and liver tissues of Göttingen minipigs. In summary, the eMIRNA pipeline presented in the current work allowed us to expand the catalogue of porcine miRNAs and showed better performance than other commonly used miRNA prediction approaches. More importantly, the flexibility of our pipeline makes possible its application in other yet poorly annotated non-model species.

尽管可用的microRNA（miRNA）预测工具种类繁多，但它们在家庭物种中发现和注释新型miRNA基因的应用仍然受到限制。在这项研究中，我们设计了一条全面的流水线（eMIRNA），用于在尚未注释的猪基因组中进行miRNA鉴定，并证明了实施基序搜索位置细化策略以准确确定前体miRNA边界的有用性。中臀小RNA片段对48只杜洛克小母猪的骨骼肌进行测序，并用于预测新的miRNA基因座。此外，我们选择了人类miRNA注释，用于基于同源性的猪miRNA与人类基因组中直系同源基因的搜索。在猪肌肉转录组中鉴定出总共20种新颖表达的miRNA，并且使用人miRNA注释通过基于同源性的搜索还检测到27种其他新颖的猪miRNA。存在三种选定的新型miRNA（ssc-miR-483，ssc-miR484和ssc-miR-200a）通过哥廷根小型猪的肌肉和肝脏组织中的逆转录定量实时PCR分析进一步证实。总而言之，当前工作中介绍的eMIRNA管线使我们能够扩展猪miRNA的目录，并显示出比其他常用的miRNA预测方法更好的性能。更重要的是，我们管道的灵活性使其有可能在其他尚未注释的非模型物种中应用。