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. 2023 Dec 1:(202):10.3791/65779.
doi: 10.3791/65779.

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

Sreenivas Eadara  1 , Xinbei Li  1 , Emily A Eiss  1 , Mollie K Meffert  2
Affiliations

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

Sreenivas Eadara et al. J Vis Exp. .

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.

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Figures

Figure 1:
Figure 1:. Formatting for data directories.
Files containing raw reads for each sequencing library must be provided in the .fastq.gz format. (A) If the libraries are not paired-end, a single .fastq.gz file will be used in analysis. This file should be named ‘SAMPLE.fastq.gz’ where SAMPLE is the exact sample name provided by the user in the adapter file. The file should be contained within a folder matching the sample name exactly. (B) For paired-end sequencing libraries, two .fastq.gz files will be used. These files should be named ‘SAMPLE-R1.fastq.gz’ and ‘SAMPLE-R2.fastq.gz’ and should be located within a folder matching the sample name exactly. All such directories named SAMPLE should be located within the same parent directory, which the user will provide to SCRAP as the “sample directory”.
Figure 2:
Figure 2:. Proportion of miRNA:target RNA interactions by Target Type and Peak Calling methods.
Chimeric sncRNA:target RNA sequencing published data from libraries prepared using CLEAR-CLIP (SRR2413277 - SRR2413295) were analyzed using a modified version of SCRAP (SCRAP release 2.0) with rRNA filtering implemented. Pre-miRNAs, tRNAs, and rRNAs were filtered, and distinct peak calling settings were used for ‘high-confidence’ (minimum 3 reads and 2 libraries) and ‘all interactions’ (minimum 1 read and 1 library). Interactions were grouped by miRNA family or ungrouped. Relative fractions of chimeric RNA reads for the categories (CDS, 5’ UTR, intergenic, intron, 3’UTR) were calculated and graphed.
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