Edi-Set-Flow - Detecting RNA editing at scale

A robust pipeline for RNA editing detection and differential analysis in bulk RNA-seq

This is a beta release - please try it and report any issues here

Overview

Getting Started

1) Requirements

• Nextflow (≥ 22.03.0)
• Container engine, any of:
  • Apptainer
  • Singularity
  • Docker

2) Sample Manifest (CSV format)

• Required Columns:
  • "sample_id" - uniuqe identified for sample
  • "fastq1" - path to first fastq file
  • "fastq2" - path to second fastq file (if paired data)
  • "group" - experimental condition of interest
• Optional Columns:
  • Arbitrary covariates to be include in GLM as fixed effects
  • Note: Columns with numbers will be interpreted as numeric by the GLM, otherwise they will be treated as factors
• Example:

  sample_id,fastq1,fastq2,group,sex,age
  SRR1311086,/PATH/TO/SRR1311086_1.fastq.gz,/PATH/TO/SRR1311086_2.fastq.gz,cortex,male,50
  SRR1477080,/PATH/TO/SRR1477080_1.fastq.gz,/PATH/TO/SRR1477080_2.fastq.gz,cerebellum,female,60
  SRR1085825,/PATH/TO/SRR1085825_1.fastq.gz,/PATH/TO/SRR1085825_2.fastq.gz,hippocampus,male,50
  ...
  

3) Run Edi-Set-Flow

• Example:

  nextflow run bahlolab/Edi-Set-Flow \
      -revision 25.08-beta.1\
      -profile hg38,singularity \
      -resume \
      --input sample_manifest.csv \
      --report_fixed_effects sex,age \
      --outdir esf_results
  

• Notes:
  • Profiles:
    • Genome: 'hg38', 'mm10' or 'mm39' are supported. Others genome builds require custom specification, see nextflow.config
    • Container Engine: 'singularity', 'apptainer', or 'docker'
    • Examples: -profile hg38,singularity or -profile mm10,docker
  • Resources (e.g. reference genome) are downloaded and stored in directory 'esf_resources', use --resource_dir /path/to/resource_dir to override and share between runs
  • REDIPortal annotation is only supported for 'hg38'

Output Files

File	Description
EdiSetFlow.report.html	Interactive Edi-Set-Flow HTML report — see example (GTEx brain)
EdiSetFlow.sample_counts.csv.gz	Reference and alternate allele counts per site and sample
EdiSetFlow.sample_summary.csv.gz	Summary statistics (median depth & editing rate) per sample
EdiSetFlow.site_summary.csv.gz	Per-site statistics and annotation (e.g. gene, region, REDIPORTAL, consequence)
EdiSetFlow.glm_summary.csv.gz	GLM coefficients, standard errors, and significance per site
EdiSetFlow.glm_anova.csv.gz	ANOVA test results for each model term per site
EdiSetFlow.glm_contrasts.csv.gz	Pairwise group comparisons for differential editing
EdiSetFlow.glm_margins.csv.gz	Estimated marginal editing rates per group
multiqc_report.html	MultiQC summary report (fastp, STAR, mosdepth, etc.)

References

Piechotta, M., Naarmann-de Vries, I. S., Wang, Q., Altmüller, J. & Dieterich, C. (2022)
RNA modification mapping with JACUSA2.
Genome Biology, 23(1), 115.

D’Addabbo, P., Cohen-Fultheim, R., Twersky, I., Fonzino, A., Silvestris, D. A., Prakash, A., … & Picardi, E. (2025)
REDIportal: toward an integrated view of the A-to-I editing.
Nucleic Acids Research, 53(D1), D233–D242.

McLaren, W., Gil, L., Hunt, S. E., Riat, H. S., Ritchie, G. R., Thormann, A., … & Cunningham, F. (2016)
The Ensembl Variant Effect Predictor.
Genome Biology, 17, 1–14.

da Veiga Leprevost, F., Grüning, B. A., Alves Aflitos, S., Röst, H. L., Uszkoreit, J., Barsnes, H., … & Perez-Riverol, Y. (2017)
BioContainers: an open-source and community-driven framework for software standardization.
Bioinformatics, 33(16), 2580–2582.

Di Tommaso, P., Chatzou, M., Floden, E. W., Barja, P. P., Palumbo, E. & Notredame, C. (2017)
Nextflow enables reproducible computational workflows.
Nature Biotechnology, 35(4), 316–319.