Identification and annotation of small RNA genes using ShortStack

Abstract

Highly parallel sequencing of cDNA derived from endogenous small RNAs (small RNA-seq) is a key method that has accelerated understanding of regulatory small RNAs in eukaryotes. Eukaryotic regulatory small RNAs, which include microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs), typically derive from the processing of longer precursor RNAs. Alignment of small RNA-seq data to a reference genome allows the inference of the longer precursor and thus the annotation of small RNA producing genes. ShortStack is a program that was developed to comprehensively analyze reference-aligned small RNA-seq data, and output detailed and useful annotations of the causal small RNA-producing genes. Here, we provide a step-by-step tutorial of ShortStack usage with the goal of introducing new users to the software and pointing out some common pitfalls.

Keywords: Bioinformatics; Genome annotation; High-throughput sequencing; Small RNA; microRNA; siRNA.

Figure 1

Inputs and outputs of ShortStack. A. Schematic showing possible inputs and outputs of a ShortStack analysis. Letters in colored circles indicate different types of files or data. B. Required inputs and their outputs for analysis. Three possible input combinations are possible, depending on the requirements for adapter trimming and alignment. Letters in colored circles refer to file/data types in panel A. C. Optional inputs. Letters in colored circles refer to file/data types in panel A.