High-precision Detection of RNA Editing Sites using Calibrated Differential RNA Editing Scanner

Summary

This protocol describes the use of the Calibrated Differential RNA Editing Scanner (CADRES), a computational workflow that integrates DNA–RNA joint variant calling, signal‑optimized recalibration, and replicate‑aware statistical modeling to identify differential RNA editing sites with high precision.

Abstract

Accurate delineation of RNA editing remains technically challenging because genuine post‑transcriptional alterations must be distinguished from genomic variants and sequencing artifacts. This difficulty is especially marked for cytidine‑to‑uridine editing catalyzed by APOBEC enzymes, where intermixed DNA and RNA changes obscure the true editing signal. The Calibrated Differential RNA Editing Scanner (CADRES) provides a structured computational framework to address these limitations through integrated DNA–RNA variant interrogation and targeted preservation of authentic editing signatures. This protocol presents the CADRES workflow, including data preparation, joint RNA‑variant calling, signal-preserving base-quality recalibration, artifact filtering, and differential assessment of RNA editing between experimental conditions. CADRES supports paired RNA‑seq and whole‑genome or whole‑exome sequencing with biological replication.  A multi-stage filtering strategy, including homopolymer removal and PBLAT-based paralogue screening, systematically reduces false positives while preserving low-frequency editing events. By combining calibration with replicate-aware modeling, CADRES increases the precision and reproducibility of RNA editing analysis, enabling interrogation of editing dynamics across diverse biological contexts. Compared with established methods, CADRES is designed to improve precision in RNA editing detection, particularly for APOBEC-mediated C-to-U events.

Introduction

RNA editing constitutes a dynamic layer of post‑transcriptional regulation that enables site‑specific nucleotide substitutions within RNA transcripts without altering the underlying DNA sequence. In metazoans, adenosine‑to‑inosine (A>I) deamination mediated by ADAR enzymes is the predominant form and contributes to transcript diversification, mRNA stability, innate immune modulation, and neuronal function^1,2. Cytidine‑to‑uridine (C>U) (hereafter "C>U" in biological context; "C>T" in sequencing context) deamination, catalyzed b....

Protocol

This protocol describes a purely computational bioinformatics workflow for identifying C>U RNA editing events using the CADRES framework. All steps are performed within a Linux environment using the command line. Only publicly available sequencing datasets are used, and no human or vertebrate subjects are involved.

1. Environment setup and software installation

NOTE: Minimum computational requirements for the CADRES workflow are as follows: CPU ≥ 8 cores (16 cores recommended), RAM ≥ 32 GB (64 GB recommended for whole-genome datasets), and disk space ≥ 100 GB.

1. Co....

Results

To evaluate CADRES under realistic experimental conditions, we used an inducible APOBEC3B (A3B) system in 293T cells. A doxycycline‑responsive lentiviral construct expressing A3B-GFP was introduced into 293T cells, and stable integrants were selected with puromycin. Induction with doxycycline for 72 h produced robust A3B-GFP expression, confirmed by GFP fluorescence and increased A3B mRNA levels. Matched induced and non‑induced samples were then subjected to uniform DNA and RNA extraction, library preparation, and sequen.......

Discussion

The CADRES workflow presented here provides a calibrated, internally consistent strategy for detecting differential RNA editing events with high specificity, particularly C>U deamination catalyzed by APOBEC enzymes. Several steps within the protocol are pivotal to its accuracy. Matched genomic and transcriptomic sequencing is essential for distinguishing genuine RNA edits from underlying DNA polymorphisms, while the boost recalibration procedure safeguards authentic RNA variants from being erroneously penalized durin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCFtools	Samtools project	N/A	Version 1.21. Variant calling and VCF manipulation. URL: https://github.com/samtools/bcftools
Bedtools	Quinlan Lab	N/A	Version 2.31.1. Genome arithmetic operations. URL: https://github.com/arq5x/bedtools2
Biopython	Biopython project	N/A	Version 1.85. Python tools for molecular biology. URL: https://www.biopython.org
BWA-MEM	GitHub (lh3/bwa)	N/A	Version 0.7.18. DNA-seq alignment. URL: https://github.com/lh3/bwa
CADRES source code	GitHub (junsun-hash/CADRES)	N/A	Version 1.0.0. CADRES pipeline scripts. URL: https://github.com/junsun-hash/CADRES
Conda or Miniconda	Anaconda Inc.	N/A	Version 23.1. Package and environment manager. URL: https://docs.conda.io/en/latest/miniconda.html
dbSNP GRCh38 VCF	NCBI	N/A	Build 155. Common germline variants database. URL: https://ftp.ncbi.nih.gov/snp/
GATK4	Broad Institute	N/A	Version 4.3.0.0. Genome Analysis Toolkit. URL: https://github.com/broadinstitute/gatk
Git	Software Freedom Conservancy	N/A	Version 2.39. Version control system. URL: https://git-scm.com
gnomAD GRCh38 VCF	Broad Institute	N/A	Version 3.1. Population allele frequencies. URL: https://gnomad.broadinstitute.org
GTF annotation file	Ensembl	N/A	Release 109. Gene annotation for GRCh38. URL: https://www.ensembl.org
Human reference genome GRCh38	Ensembl/UCSC	N/A	Release 109. Reference genome assembly. URL: https://www.ensembl.org or https://hgdownload.soe.ucsc.edu
Linux workstation or server	Various	N/A	Ubuntu 20.04. x86_64 architecture required. URL: https://ubuntu.com
pblat	UCSC Genome Browser	N/A	Version 2.5.1. Parallel BLAT realignment. URL: https://github.com/ucscGenomeBrowser/kent
Picard	Broad Institute	N/A	Version 2.20.8. NGS data manipulation. URL: https://github.com/broadinstitute/picard
Python	Python Software Foundation	N/A	Version 3.9.19. Programming language. URL: https://www.python.org
R	R Foundation	N/A	Version 4.5.2. Statistical computing. URL: https://www.r-project.org
R package: forcats	CRAN	N/A	Version 1.0.0. Factor manipulation. URL: https://cran.r-project.org/package=forcats
R package: ggplot2	CRAN	N/A	Version 4.0.1. Data visualization. URL: https://cran.r-project.org/package=ggplot2
R package: ggrepel	CRAN	N/A	Version 0.9.5. Text label repulsion. URL: https://cran.r-project.org/package=ggrepel
R package: lme4	CRAN	N/A	Version 1.1.35. Linear mixed-effects models. URL: https://cran.r-project.org/package=lme4
R package: readr	CRAN	N/A	Version 2.1.5. Fast file reading. URL: https://cran.r-project.org/package=readr
R package: stringr	CRAN	N/A	Version 1.6.0. String manipulation. URL: https://cran.r-project.org/package=stringr
REDIportal reference	University of Bologna	N/A	Version 2.0. A-to-I RNA editing sites database. URL: http://srv00.recas.ba.infn.it/atlas/
RefGene annotation	UCSC Table Browser	N/A	Release 109. Gene structure annotation. URL: https://genome.ucsc.edu/cgi-bin/hgTables
Samtools	Samtools project	N/A	Version 1.21. BAM file manipulation. URL: https://github.com/samtools/samtools
STAR aligner	GitHub (alexdobin/STAR)	N/A	Version 2.7.11b. RNA-seq alignment. URL: https://github.com/alexdobin/STAR