Identification and annotation of small RNA genes using ShortStack

Saima Shahid, Michael J. Axtell

Research output: Contribution to journalArticlepeer-review

41 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)20-27
Number of pages8
Issue number1
StatePublished - May 1 2014

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • General Biochemistry, Genetics and Molecular Biology


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