![]() In this article, we assume that the reader has a basic working knowledge of the Bourne Again Shell (BASH) or similar shell applications on their system. ShortStack is a command-line program for Linux or Mac OSX operating systems. ![]() Factors affecting analysis and detection sensitivity in different stages of the computational pipeline are addressed. In this article, we provide a step-by-step tutorial of ShortStack usage for the current version 1.1.0 ( Fig. Based on the small RNA alignment patterns, ShortStack identifies and annotates both MIRNA and non- MIRNA loci, and provides detailed descriptions of the small RNA populations emanating from each locus. These improvements are layered on top of the core annotation and quantification methods that were previously described. Additionally, ShortStack also can now accept multiple small RNA-seq libraries in a single run. The current version (1.1.0) now is able to handle adapter-trimming and alignment of small RNA-seq data prior to annotation and quantification ( Fig. Since the publication of the first ShortStack paper, based on version 0.4.0 of the software, the capabilities and performance of ShortStack have been substantially improved. With this need in view, ShortStack was developed for comprehensive analysis of small RNA-seq data. Thus, there is an emerging need for computational tools that can provide global annotations of both MIRNA and non- MIRNA loci from small RNA-seq datasets. Unlike MIRNA loci, the loci producing other types of endogenous regulatory small RNAs have had little systematic curation. However, miRNAs frequently comprise only a small percentage of the entire small RNA repertoire, especially in plants and in animal germ-line associated tissues. ![]() ![]() MIRNA loci, which produce the primary stem-loop transcripts that are processed to yield mature microRNAs (miRNAs), represent the best characterized type of small RNA gene they are readily discerned from genome-aligned small RNA-seq data, and are extensively annotated in multiple species by the miRBase database. Highly parallel sequencing of cDNAs derived from small RNAs (small RNA-seq) allows comprehensive assessment of regulatory small RNA accumulation, especially when combined with alignments to a reference genome. Rapid progress in high throughout sequencing methodologies has played a key role in unveiling the scope of regulatory small RNAs in eukaryotes. ![]()
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