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 https://github.com/MikeAxtell/ShortStack/releases/
Test data for ShortStack are also available at http://axtelldata.bio.psu.edu/data/ShortStack_TestData/
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