To overcome these issues, we have developed a new software, named DROPA (DRIP-seq Optimized Peak Annotator), which makes use of gene expression data to annotate R-loop peaks to strand templates and expressed genes. As R-loops can span several gene features (REFs), the assignment of R-loop peaks to a unique feature may not be appropriate. Nevertheless, DRIP peaks are markedly different from traditional ChIP (Chromatin Immunoprecipitation) peaks of transcription factors as the former peaks are usually much longer than the latter ones, spanning across several genes features or different genes. Moreover, DRIP-seq data are commonly analyzed with standard peak callers, such as MACS (Model-based Analysis of ChIP-Seq), to identify regions with above-threshold coverage signals, usually called “peaks”. G-quadruplexes) or with basic directional mechanisms such as replication and transcription.
Understanding the DNA strand forming the hybrid is essential to investigate the dynamic interplay of R-loops with other nucleic acid structure (e.g. However, the most common technique (DRIP) allows the detection of R-loop regions without providing the strand information of the DNA:RNA hybrid. Immunoprecipitation-based techniques, generally known as DRIP (DNA:RNA Immunoprecipitation), coupled with parallel sequencing (DRIP-seq), are widely used to maps R-loops genome-wide and several DRIP variants have been developed with the intent to improve the identification of genomic R-loops. Genome-wide maps of these peculiar nucleic acid structures have boosted our understanding of R-loop biology. However unscheduled R-loop formation is associated with DNA damage accumulation, genome instability and genetic diseases. R-loops have been shown to be involved in many nuclear processes such as transcription regulation, DNA methylation modulation and DNA repair mechanisms. R-loops form co-transcriptionally when nascent RNAs anneal back to DNA template strand. R-loops are three stranded nucleic acid structures composed by a DNA:RNA hybrid duplex and a displaced ssDNA (single strand DNA) strand. Its output can easily be integrated into pipelines to perform downstream analyses, while useful and informative summary plots and statistical enrichment tests can be produced. ConclusionsĭROPA is a fully customizable peak-annotation tool optimized for co-transcriptional DRIP-seq peaks, which allows a finest gene annotation based on gene expression information.
A comparison of DROPA performance with three widely used annotation tools show that it identifies less false positive annotations than the others.
DROPA allows to assign R-loop peaks to the DNA template strand in gene body with a false positive rate of less than 7%.
#Homer annotate peaks full
DROPA allows a full customization of annotation options, ranging from the choice of reference datasets to gene feature definitions. Here, we introduce DRIP-seq Optimized Peak Annotator (DROPA), a new tool for gene annotation of R-loop peaks based on gene expression information. However, annotation of DRIP-seq peaks to genes can be a tricky step, due to the lack of strand information when using the common basic DRIP technique. DRIP (DNA:RNA Immunoprecipitation)-seq techniques are widely used to map R-loops genome-wide providing insights into R-loop biology. R-loops are three-stranded nucleic acid structures that usually form during transcription and that may lead to gene regulation or genome instability.
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