Overview

RNAchrom is a comprehensive and flexible Nextflow pipeline designed to process RNA-DNA interactome sequencing data. It efficiently handles large-scale data from various experiments such as GRID-seq, RADICL-seq, and iMARGI, providing a streamlined workflow for analyzing RNA-DNA interactions.

The typical RNAchrom pipeline involves stages such as data trimming, alignment, and annotation. The interactions between RNA and DNA components are extracted, filtered, and then visualized or further analyzed.

Key Features

• Input Management: Automatically validates input files and stages them for further processing.

• Modular Design: Includes flexible modules for deduplication, trimming, alignment, and more.

• Comprehensive Analysis: Handles both one-to-all (e.g., RAP) and all-to-all (e.g., GRID-seq) experimental designs.

• Quality Control: Integrates with FastQC and MultiQC for comprehensive quality assessments.

• Flexible Customization: Users can skip stages or customize configurations specific to their experimental setup.

Pipeline Modules

RNAchrom includes a variety of modules that can be utilized as needed in the workflow:

Module	Description
INPUT_CHECK	Validates and stages input samples.
DEDUP	Deduplicates sequencing reads.
TRIM	Trims sequencing reads to remove adaptors and low-quality bases.
ALIGN	Aligns reads to the reference genome.
ANNOTATE	Conducts annotation voting for the DNA segments.
MACS2	Identifies significant peaks in RNA-DNA interaction data.
MULTIQC	Aggregates results across multiple samples for comparative analysis.

Contents:

• Setup and Installation
  • Prerequisites
  • Installing Nextflow
  • Pipeline Dependencies
  • Installation Process
  • Running the Pipeline
  • Troubleshooting
• Input file preparation
  • Samplesheet
  • Genome
  • Gene Annotation
  • Requirements for Bed-like Gene Annotation File
  • Shared Gene Annotation Issues
• RNAchrom Configuration
  • Input Options
  • Processing Tools
  • Reference Genome
  • Annotation Files
  • Restriction Sites & Bridge Processing
  • Other Options
  • Command Flags
  • Environment Variables
  • Included Configs
• Stages of Data Analysis
  • 1. Input and Preprocessing
  • 2. Deduplication (optional)
  • 3. Trimming
  • 4. Bridge Processing (for specific experiment types)
  • 5. Restriction sites filtering
  • 6. Alignment
  • 6. Post-alignment Processing
  • 7. Contact Generation
  • 8. CIGAR Filtering (optional)
  • 9. Merging Replicates
  • 10. Chromosome Splitting (optional)
  • 11. Annotation and Voting
  • 12. Background Model Generation
  • 13. Normalization
  • 14. Peak Calling (for One-to-All experiments)
  • 15. Statistics and Visualization
  • 16. MultiQC Report
  • Main Results
• Results
  • 1. Input and Preprocessing
  • 2. Deduplication (optional)
  • 3. Trimming
  • 4. Bridge Processing (for specific experiment types)
  • 5. Alignment
  • 6. Post-alignment Processing
  • 7. Contact Generation
  • 8. CIGAR Filtering (optional)
  • 9. Merging Replicates
  • 10. Chromosome Splitting (optional)
  • 11. Annotation and Voting
  • 12. Background Model Generation
  • 13. Normalization
  • 14. Peak Calling (for One-to-All experiments)
  • 15. Statistics and Visualization
  • 16. MultiQC Report
  • Main Results

Stages

• RNAchrom pipeline walkthrough

Optional Stages

• ucaRNA assembly
• Chromatin potential

Setup and Configuration

The pipeline can be configured using custom parameter settings to fit the needs of different experimental designs. Key configuration settings include:

• Genome and Annotation: Provides support for multiple reference genomes and annotation files.

• Toolchain Configuration: Choice of tools for alignment (e.g., HISAT2, STAR) and trimming (e.g., Trimmomatic, FastP).

• Output Management: Options for generating summarized reports and logs for comprehensive analysis.

User Support and Community

• Documentation: Detailed installation and execution instructions available.

• Community Support: Engage with the community through forums and GitHub issues.

• Contributions: Open to contributions from the research community to enhance features.

For additional help and support, please check our community forums and our GitHub repository.

• Index