Figure 1.

Overview of the small RNA sequencing workflow, compared methodologies for library preparation and computational analysis pipeline.

(a) Schematic illustration of small RNA sequencing (sRNA-seq) library preparation. With exception of the SMARTer smRNA-Seq kit, all kits employed a ligation-based approach to attach 3′ and 5′ adapter to the miRNA. In the QIAseq protocol, a unique barcode is attached at the RT stage (orange dotted line), so-called unique molecular indices (UMI). Illumina adapter (purple and blue) and sequencing index (light purple) for multiplexing are added during the PCR amplification. Several steps of the protocol may introduce bias to the sequencing libraries as indicated by the yellow warning triangles. (b) Table summary of the key features of the four assessed sRNA-seq kits. (c) Small RNA sequencing data analysis pipeline. In the case of QIAseq, UMIs were trimmed alongside the adapters to allow side-by-side comparison by a single computational pipeline. Abbreviations: Nextflex Small RNA-Seq Kit v3 (Nextflex), NEBNext Small RNA Library Prep Set (NEBNext) and QIAseq miRNA Library Kit (QIAseq).