RAPID: an interactive R/Shiny platform for end-to-end 16S rRNA and ITS amplicon sequence analysis using DADA2

Abstract

MotivationAmplicon sequencing of 16S rRNA and internal transcribed spacer (ITS) gene regions is the most widely used approach for characterizing bacterial and fungal communities, respectively. The DADA2 pipeline has become a standard for inferring amplicon sequence variants (ASVs), offering single-nucleotide resolution over traditional OTU clustering. However, executing the full DADA2 workflow requires proficiency in R programming and manual coordination of multiple sequential steps, presenting a substantial barrier for researchers in clinical, environmental, and agricultural sciences who lack computational training. ResultsWe present RAPID (R-based Amplicon Pipeline for Interactive DADA2), a pair of R/Shiny applications providing complete graphical user interfaces for 16S rRNA and ITS amplicon sequence analysis. The 16S application implements a 10-step guided workflow from raw paired-end FASTQ files through quality filtering, error learning, dereplication, paired-read merging, chimera removal, taxonomy assignment (SILVA), phyloseq construction with data transformation (rarefaction, relative abundance, or CLR), interactive visualization (rarefaction curves, alpha diversity, NMDS, PCoA, taxonomic abundance), PERMANOVA, and ANCOM-BC2 differential abundance analysis. The ITS application extends this to an 11-step workflow, adding an automated primer removal step using cutadapt with support for multiple primers and length-variable amplicons, and uses the UNITE database for fungal taxonomy. Both applications feature asynchronous background processing, session persistence, real-time progress monitoring, publication-ready figure export, and comprehensive result downloads. AvailabilityRAPID is freely available at https://github.com/beantkapoor786/RAPID. Both applications can be installed locally on any system with R (version 4.0 or higher) and run as local web applications accessible through a standard browser.