RNAcontacts, a pipeline for predicting contacts from RNA proximity ligation assays

Abstract

Background High-throughput RNA proximity ligation assays are molecular methods that simultaneously analyze spatial proximity of many RNAs in living cells. Their principle is based on cross-linking, fragmentation, and consequent religation of RNAs followed by high-throughput sequencing. The generated fragments have two distinct types of splits, one resulting from pre-mRNA splicing, and the other resulting from ligating spatially close RNA strands. Findings Here, we present RNAcontacts, a universal pipeline for detecting RNA-RNA contacts in high-throughput RNA proximity ligation assays. It circumvents the inherent problem of mapping sequences with two distinct split types using a two-pass alignment, in which splice junctions are inferred from a control RNA-seq experiment on the first pass and then provided to the aligner on the second pass as bona fide introns. This approach allows for a more sensitive detection of RNA contacts and has higher specificity with respect to splice junctions that are present in the biological sample in comparison to previously developed methods. RNAcontacts extracts contacts, clusters their ligation points, computes the read support, and generates tracks for the visualization through the UCSC Genome Browser. It is implemented in a reproducible and scalable workflow management system Snakemake that allows fast and uniform processing of multiple datasets. Conclusions RNAcontacts represents a generic pipeline for the detection of RNA contacts that can be used with any proximity ligation method as long as one of the interacting partners is RNA. RNAcontacts is available via github at https://github.com/smargasyuk/RNAcontacts/