2021 FDA Science Forum
Toolkit for Quick and Low-Cost Differential Expression Analysis of RNA-Seq Data
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Contributing OfficeCenter for Veterinary Medicine
Abstract
Bulk RNA sequencing (RNA-seq) is a powerful and widely used tool to identify biomarkers. The most common RNA-seq analysis is differential expression (DE) combined with geneset enrichment analysis (GSEA), for which there are well-benchmarked analysis tools available. However, these tools are relatively inaccessible to scientists without bioinformatics expertise. Thus, we sought to create an end-to-end toolkit for DE analysis that could be quickly employed by all FDA scientists, using existing resources and with minimal cost to the user.
Our toolkit makes use of two user-friendly, open-source interfaces: Galaxy and RStudio. First, precisionFDA or CDRH's Galaxy is used to trim the sequencing reads (trimmomatic), assess data quality (FastQC), align (HISAT2), and generate gene expression counts (featureCounts). Then, RStudio software is used to identify DE genes (edgeR) and GSEA (clusterProfiler). Finally, RStudio’s Shiny interactive dashboards are used to explore and visualize the DE and GSEA.
The entire analysis can be performed on a laptop, and typical analyses (6-12 samples) can be completed within one day. CDRH usage is low cost ($5 per CPU day) and Galaxy runs in a web browser without additional cost. RStudio software is free and can be run on PC, Mac, and Linux computers. The Shiny dashboards allows users to generate custom, publication quality figures and tables without costly bioinformatics support.
We have created a toolkit for FDA research and review scientists to perform RNA-seq analysis without the need for bioinformatics expertise. The analysis is low cost, quick, reproducible, and available to FDA scientists to support regulatory research and evaluation.
