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

Abstract

We recently developed the rG4-seq method to detect and map in vitro 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 ( https://github.com/TF-Chan-Lab/rG4-seeker ), 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.

G-quadruplex RNA motifs influence gene expression in the malaria parasitePlasmodium falciparum

“…These imperfections do not necessarily compromise rG4 folding but they have been suggested to have a negative impact on the thermostability of the resultant rG4 structures 18,25 . The P. falciparum transcriptome also harboured many guanine-rich potential rG4s and RNA G-triplex (rG3) motifs that could potentially fold into quadruplex/triplex structures despite their nucleotide sequences not matching the four better-established structural motif definitions 26 .…”

“…Reverse transcriptase stalling (RTS) site analysis and rG4 calling were conducted using the rG4-seeker pipeline 43 . The definition of rG4 structural motifs has been previously described 18,43 . The intersection and union of detected rG4s across the four rG4-seq experiments (2 replicates, 2 conditions) were computed based on the overlapping of genomic coordinates of the rG4 sequences.…”

“…Pre-processing and short read alignment of rG4-seq sequencing data were conducted as previously described 43 . Reverse transcriptase stalling (RTS) site analysis and rG4 calling were conducted using the rG4-seeker pipeline 43 . The definition of rG4 structural motifs has been previously described 18,43 .…”

G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum

“…Pre-processing and short read alignment of sequencing data were conducted as previously described ( 28 ). Reverse transcriptase stalling (RTS) site analysis and rG4 calling were conducted using the rG4-seeker pipeline ( 28 ). The definition of rG4 structural motifs has been previously described ( 18 , 28 ).…”

“…Reverse transcriptase stalling (RTS) site analysis and rG4 calling were conducted using the rG4-seeker pipeline ( 28 ). The definition of rG4 structural motifs has been previously described ( 18 , 28 ). In brief, sequencing reads from rG4-stabilizing conditions (K + and K + /PDS) were compared to those from non-rG4-stabilizing condition (Li + ) to detect RTS sites specific to the rG4-stabilizing conditions, resulting in four sets of RTS sites detections (two replicates, two rG4-stabilizing conditions).…”