Computational Analysis Tutorial for Chimeric Small Noncoding RNA: Target RNA Sequencing Libraries

Summary

Here, we present a protocol demonstrating the installation and use of a bioinformatics pipeline to analyze chimeric RNA sequencing data used in the study of in vivo RNA:RNA interactions.

Abstract

An understanding of the in vivo gene regulatory interactions of small noncoding RNAs (sncRNAs), such as microRNAs (miRNAs), with their target RNAs has been advanced in recent years by biochemical approaches which use cross-linking followed by ligation to capture sncRNA:target RNA interactions through the formation of chimeric RNAs and subsequent sequencing libraries. While datasets from chimeric RNA sequencing provide genome-wide and substantially less ambiguous input than miRNA prediction software, distilling this data into meaningful and actionable information requires additional analyses and may dissuade investigators lacking a computational background. This report provides a tutorial to support entry-level computational biologists in installing and applying a recent open-source software tool: Small Chimeric RNA Analysis Pipeline (SCRAP). Platform requirements, updates, and an explanation of pipeline steps and manipulation of key user-input variables is provided. Reducing a barrier for biologists to gain insights from chimeric RNA sequencing approaches has the potential to springboard discovery-based investigations of regulatory sncRNA:target RNA interactions in multiple biological contexts.

Introduction

Small noncoding RNAs are highly studied for their post-transcriptional roles in coordinating expression from suites of genes in diverse processes such as differentiation and development, signal processing, and disease^1,2,3. The ability to accurately determine the target transcripts of gene-regulatory small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), is of importance to studies of RNA biology at both basic and translational levels. Bioinformatic algorithms that exploit anticipated complementarity between the miRNA seed sequence and its potential targets have been f....

Protocol

NOTE: The protocol will begin with downloading and installing software required to analyze chimeric RNA sequencing libraries using SCRAP.

1. Installation

1. Before installing SCRAP, install the dependencies Git and Miniconda on the machine to be used for the analyses. Git is likely already installed. On the Mac OSX platform, for example, verify this using which git to see that the "git" utility is present and installed in this directory. Check if Miniconda is installed using which conda. If nothing is returned, install Miniconda. Miniconda requires 400 MB of disk space to i....

Results

Results for sncRNA:target RNA detected by a modified version of SCRAP (SCRAP release 2.0, which implements modifications for rRNA filtering) on previously published sequencing datasets prepared using CLEAR-CLIP⁹ is shown in Figure 2 and Table 1. Users can appreciate the decrease in the relative fraction miRNA interactions with intron regions which occurs following the isolation of high-confidence interactions by peak calling in SCRAP. Additional data .......

Discussion

This protocol on the use of SCRAP pipeline for analysis of sncRNA:target RNA interactions is designed to assist investigators who are entering into computational analysis. Completion of the tutorial is expected to guide investigators with entry-level or greater computational experience through the steps required for installation and use of this pipeline and its application to analyze data gained from chimeric RNA sequencing libraries. Steps critical to the completion of this protocol include correct reference installatio.......

Materials

Name	Company	Catalog Number	Comments
Genomes	UCSC Genome browser	N/A	https://genome.ucsc.edu/ or https://www.ncbi.nlm.nih.gov/data-hub/genome/
Linux	Linux	Ubuntu 20.04 or 22.04 LTS recommended
Mac	Apple	Mac OSX (>11)
Platform setup	GitHub	N/A	https://github.com/Meffert-Lab/SCRAP/blob/main/PLATFORM-SETUP.md]
SCRAP pipeline	GitHub	N/A	https://github.com/Meffert-Lab/SCRAP
Unix shell	Unix operating system	bash >=5.0
Unix shell	Unix operating system	zsh (5.9 recommended)
Windows	Windows	WSL Ubuntu 20.04 or 22.04 LTS