rnaCrosslinkOO: an object-oriented R package for the analysis of RNA structural data generated by RNA crosslinking experiments

Abstract

Summary: RNA (ribonucleic acid) molecules have secondary and tertiary structures in vivo which play a crucial role in cellular processes such as the regulation of gene expression, RNA processing and localization. The ability to investigate these structures will enhance our understanding of their function and contribute to the diagnosis and treatment of diseases caused by RNA dysregulation. However, there are no mature pipelines or packages for processing and analyzing complex in vivo RNA structural data. Here, we present rnaCrosslinkOO (RNA Crosslink Object-Oriented), a novel software package for the comprehensive analysis of data derived from the COMRADES (Crosslinking of Matched RNA and Deep Sequencing) method. rnaCrosslinkOO offers a comprehensive pipeline from raw sequencing reads to the identification and comparison of RNA structural features. It includes read processing and alignment, clustering of duplexes, data exploration, folding and comparisons of RNA structures. rnaCrosslinkOO also enables comparisons between conditions, the identification of inter-RNA interactions, and the incorporation of reactivity data to improve structure prediction.

Availability and implementation: rnaCrosslinkOO is freely available to noncommercial users and implemented in R, with the source code and documentation accessible at https://CRAN.R-project.org/package=rnaCrosslinkOO. The software is supported on Linux, macOS, and Windows platforms.

Figure 1.

rnaCrosslinkOO. Main steps of the algorithm are shown in a,b, and c and optional analysis steps are shown in d-i. (a) Input for rnaCrosslinkOO and the main slots of the rnaCrosslinkDataSet object displayed with grey boxes with the functions used to access the slots in green. sampleTable—A table with the following column names; file (file path for sample), group (a code for the sample group), sample (a numeric value showing which replicates belong to which group), sampleName (a unique sample identifier). inputFiles, matrixList—The inputFiles slot contains the duplexes in their original input format (Supplementary Table S3) and the matrixList contains the data as contact matrices. clusterGRangesList, ClusterTableList—These two slots are related to the clustering of the duplexes and contain a GRanges object and data frame of the cluster coordinates. viennaStructures, dgs, clusterTableFolded, and interactionTable—These slots relate to the folding of the RNA. The viennaStructures and dgs slots contain the vienna format structures and the free energy value for the predicted structures for each sample. The interactionTable contains the constraints identified from folding each of the clusters and the clusterTableFolded slot contains the predicted fold for each cluster. (a–i) Schematics and examples of steps in the rnaCrosslinkOO package, further detail for each step can be found in the main text.