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NGStoolkit is a one-stop shop next-generation sequencing analysis toolkit in Bash that allows you to run a complete NGS analysis pipeline (e.g., RNA-seq) by issuing just one command from the Terminal. Currently, NGStoolkit supports RNA-seq analysis via a single-click automated RNA-seq pipeline starting with bz2 files through differential expression.

There are 6 input parameters:

  • Pipeline type { simple, complete }. simple is for individual flow cells where all people want are the stats for a given run, and complete is through from bz2 to differential expression.
  • Genome type { hg19, hg38, mm10, rn6 }. Specify the genome for the samples.
  • Read type { paired, single }. Paired/single ended read flag.
  • Configuration file { <filename> }. This file is a list of sample IDs and the groups for differential expression. First column is the sample ID and the second column is the group.
  • Stranded information { u, s, r }. Type of strandedness to take the counts from STAR output. u = unstranded, s = stranded, r = reverse stranded as defined by STAR.


sh <pipeline_type> <genome> <paired/single> <config> <strandedness>

Future Plans

Current work is underway to expand NGStoolkit into ChIP-seq, methyl-seq, and exome-seq analysis territory. We are also expanding from a hardcoded HPC environment to a flexible cloud infrastructure.


NGStoolkit is an ongoing bioinformatics software project financially supported by the United States Department of Defense (DoD) through the National Defense Science and Engineering Graduate Fellowship (NDSEG) Program. This research was conducted with Government support under and awarded by DoD, Army Research Office (ARO), National Defense Science and Engineering Graduate (NDSEG) Fellowship, 32 CFR 168a.