REVERSE: a user-friendly web server for analyzing next-generation sequencing data from in vitro selection/evolution experiments

Next-generation sequencing (NGS) enables the identification of functional nucleic acid sequences from in vitro selection/evolution experiments and illuminates the evolutionary process at single-nucleotide resolution. However, analyzing the vast output from NGS can be daunting, especially with limited programming skills. We developed REVERSE (Rapid EValuation of Experimental RNA Selection/Evolution) (https://www.reverseserver.org/), a web server that implements an integrated computational pipeline through a graphical user interface, which performs both pre-processing and detailed sequence level analyses within minutes. Raw FASTQ files are quality-filtered, dereplicated, and trimmed before being analyzed by either of two pipelines. The first pipeline counts, sorts, and tracks enrichment of unique sequences and user-defined sequence motifs. It also identifies mutational intermediates present in the sequence data that connect two input sequences. The second pipeline sorts similar sequences into clusters and tracks enrichment of peak sequences. It also performs nucleotide conservation analysis on the cluster of choice and generates a consensus sequence. Both pipelines generate downloadable spreadsheets and high-resolution figures. Collectively, REVERSE is a one-stop solution for the rapid analysis of NGS data obtained from in vitro selection/evolution experiments that obviates the need for computational expertise.