RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

Abstract

The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR, and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection.

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters

“…Current R-loop profiling methods appear to have an intrinsic limitation in pinpointing the exact location of R-loops in individual genomic fragments captured by S9.6 according to a recent analysis (Halász et al, 2017). Diminished signals upon RNase H digestion may reflect the absence of R-loop for capture but do not necessarily show where a specific R-loop(s) is in individual restriction fragments.…”

“…The majority of these methods use the S9.6 antibody to capture RNA/DNA hybrids from restriction digested genomic DNA followed by deep sequencing of captured DNA (DNA:RNA immunoprecipitation [DRIP] sequencing [DRIP-seq] and its derivatives) or RNA (DNA:RNA immunoprecipitation followed by cDNA conversion [DRIPc]-seq and its derivatives) (Chédin, 2016). Because of limited resolution with restriction digestion, additional efforts have also been made to use sonication to increase R-loop mapping resolution (Halász et al, 2017; Nadel et al, 2015); however, such treatment may destroy some fragile R-loops in the absence of fixation. These technical problems may thus account for various inconsistent results in the literature.…”

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast

“…The direct consequences of these observations on the accuracy of R-loop mapping by DNA:RNA immuno-precipitation (DRIP)-like methods have not yet been rigorously assessed. Preliminary RNase A treatment has been proposed to increase the specificity of R-loop mapping using DRIP approaches [13], but this was later disputed [14]. However, whether or not RNase A treatment does modulate the efficacy of DRIP, it does not help to evaluate whether dsRNAs are able to interfere with the quantitative recovery of R-loops using S9.6, as RNase A will degrade single-stranded RNA but not dsRNA.…”

In Silico Restriction Enzyme Digests to Minimize Mapping Bias in Genomic Sequencing

“…Genome fragmentation by REs is routinely used in multiple genomic mapping technologies, including RNA-DNA hybrid (R-loop) immunoprecipitation sequencing (DRIP-seq),1, 2 chromosome conformation capture (4C/5C, Hi-C), 3 reduced-representation bisulfite sequencing (RRBS), 4 and restriction site associated marker (RAD) genotyping 5 . The performance of these approaches depends on (1) the length distribution of the restriction fragments (determining the spatial resolution of the assay) and (2) the randomness of RE digestion (ensuring that all genomic regions are sampled with an equal probability) 6 .…”