rG4-seeker enables high-confidence identification of novel and non-canonical rG4 motifs from rG4-seq experiments

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>We recently developed the rG4-seq method to detect and map <jats:italic>in vitro</jats:italic> RNA G-quadruplex (rG4s) structures on a transcriptome-wide scale. rG4-seq of purified human HeLa RNA has revealed many non-canonical rG4s and the effects adjacent sequences have on rG4 formation. In this study, we aimed to improve the outcomes and false-positive discrimination in rG4-seq experiments using a bioinformatic approach. By establishing connections between rG4-seq library preparation chemistry and the underlying properties of sequencing data, we identified how to mitigate indigenous sampling errors and background noise in rG4-seq. We applied these findings to develop a novel bioinformatics pipeline named rG4-seeker(<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://github.com/TF-Chan-Lab/rG4-seeker">https://github.com/TF-Chan-Lab/rG4-seeker</jats:ext-link>), which uses tailored noise models to autonomously assess and optimize rG4 detections in a replicate-independent manner. Compared with previous methods, rG4-seeker exhibited better false-positive discrimination and improved sensitivity for non-canonical rG4s. Using rG4-seeker, we identified novel features in rG4 formation that were missed previously. rG4-seeker provides a reliable and sensitive approach for rG4-seq investigations, laying the foundations for further elucidation of rG4 biology.</jats:p>

rG4-seq 2.0: enhanced transcriptome-wide RNA G-quadruplex structure sequencing for low RNA input samples

“…To evaluate the improvements introduced by 5’ dU adapter in a realistic scenario of rG4-seq application, a two-replicate rG4-seq 1.0 library using 100 ng of RNA input and normal 5’ adapter was also constructed as a control group. All rG4-seq data was then pre-processed, deduplicated using unique molecular identifiers (UMIs), and analyzed with rG4-seeker to identify rG4-induced RTS events as previously described (Figure 3A, Table S2) [24]. The workflow enabled quantitative evaluation of the influence of RNA input amount on library complexity and rG4 identification outcome.…”

“…Uniquely aligned reads were deduplicated based on UMI information using UMI-tools[37]. Deduplicated and uniquely aligned reads were subjected to rG4-seeker pipeline [24] to identify RTS sites and rG4 motifs at default settings. Ensembl 97 gene annotation [40] was used to define the transcriptomic regions.…”

“…All rG4-seq data was then pre-processed, deduplicated using unique molecular identifiers (UMIs), and analyzed with rG4-seeker to identify rG4-induced RTS events as previously described (Figure 3A, Table S2) [24]. The workflow enabled quantitative evaluation of the influence of RNA input amount on library complexity and rG4 identification outcome.…”

“…In 2016, we introduced RNA G-quadruplex sequencing (rG4-seq) [22] for the identification of in vitro rG4 on a transcriptome-wide scale that can facilitate further exploration and study of in vivo rG4 structure and function. Recently, we have refined a few steps of the rG4-seq experimental procedures [23] and also developed a bioinformatics pipeline known as rG4-seeker [24]. To date, rG4-seq has been successfully applied to study the rG4s in human [22], plant [25], bacteria [26], and plasmodium [27].…”

Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0

“…In 2016, we introduced RNA G-quadruplex sequencing (rG4-seq) [22] for the identification of in vitro rG4 on a transcriptome-wide scale that can facilitate further exploration and study of in vivo rG4 structure and function. Recently, we have refined a few steps of the rG4-seq experimental procedures [23] and also developed a bioinformatics pipeline known as rG4-seeker [24]. To date, rG4-seq has been successfully applied to study the rG4s in human [22], plants [25], bacteria [26], and plasmodium [27].…”