ShortStack: Alignment, annotation, and quantification of small RNAs

ShortStack is a tool developed to process and analyze smallRNA-seq data with respect to a reference genome, and output a comprehensive and informative annotation of all discovered small RNA genes.  ShortStack discovers small RNA ‘clusters’ de novo, based on user-set thresholds, and annotates clusters with respect to small RNA size, orientation, and repetitiveness.  ShortStack also discovers and annotates MIRNA genes.  In addition, ShortStack includes a robust method to detect genes producing small RNAs in a phased manner.  It outputs a descriptive table of all results, useful genome browser tracks, and detailed text-based alignments of all MIRNAs.  It can also be used to quantify a set of input loci with genomic coordinates determined a priori by the user.

One important feature of ShortStack (version 3 and higher) is its alignment methodology, which was been optimized to find the most likely origin for reads with multiple possible alignment positions. In essence, ShortStack uses a local-weighting method that by default used the local densities of uniquely aligned reads to assign probabilities to multi-mapping reads. Our paper (Johnson et al. 2016 doi:10.1534/g3.116.030452) describes in detail the alignment procedure and benchmarks its performance against other methods.

ShortStack is a perl program.  Besides perl, ShortStack also requires samtools, RNAfold from the Vienna RNA Package, to execute. When used to control the alignment of small RNA data to a reference genome, ShortStack also requires gzip,  bowtie and bowtie-build.  Details can be found in the README file included with the program.

ShortStack can be obtained from github at

Test data for ShortStack are also available at

ShortStack is also a ‘public app’ in the iPlant Discovery Environment .. search for ‘ShortStack’ in the public apps!


CITATION: If you use ShortStack in your work, please cite one or more of the following:

Johnson NR, Yeoh JM, Coruh C, Axtell MJ. (2016) Improved placement of multi-mapping small RNAs. G3 doi: 10.1534/g3.116.030452

Axtell MJ. (2013) ShortStack: Comprehensive annotation and quantification of small RNA genes.  RNA 19: 740-751. PMID: 23610128 doi:10.1261/rna.035279.112

Shahid S. and Axtell MJ. (2013) Identification and annotation of small RNA genes using ShortStack. Methods PMID: 24139974 doi:10.1016/j.ymeth.2013.10.004