Small RNA Sequencing for miRNA Quantification

EC sections were available for a subset of 174 individuals (90 cases, 84 controls) included in our current study. In order to minimize potential batch effects, cases and controls were randomly distributed across sequencing plates. Quantification of the expression of the six miRNAs of interest was based on small RNA sequencing which was performed as part of another ongoing project. To this end, total RNA, including miRNA, was purified and quantified using the same methods as described above. Library preparation and subsequent sequencing were conducted at the NGS Competence Centre at IKMB institute (Kiel, Germany). Libraries were prepared using the NextFlex Small-RNA-Seq kit (PerkinElmer, Waltham, Massachusetts), according to the manufacturer’s instructions, and sequenced on a HiSeq 4000 instrument (Illumina, San Diego, California) with 1 ×50 bp reads. Sequencing adapters were trimmed from raw reads using Flexbar v3.4.0 [17 (link), 18 (link)]. Reads were mapped and miRNAs quantified against miRBase v22.1 [19 (link), 20 (link)] using miRDeep2 [21 (link)]. Data normalization was carried out using the R package DESeq2 v1.28.1 [22 ].