Identification and annotation of small RNA genes using ShortStack

Methods. 2014 May 1;67(1):20-7. doi: 10.1016/j.ymeth.2013.10.004. Epub 2013 Oct 16.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence
  • High-Throughput Nucleotide Sequencing
  • Humans
  • MicroRNAs / genetics*
  • Molecular Sequence Annotation*
  • Molecular Sequence Data
  • RNA, Small Interfering / genetics*
  • Sequence Alignment
  • Sequence Analysis, RNA
  • Software*

Substances

  • MicroRNAs
  • RNA, Small Interfering