Results

This pipeline outlines the key stages for analyzing all-to-all interactome sequencing data, focusing on the steps that lead to the final results of RNA-DNA contact pairs, RNA annotation, and significant peaks of chromatin-interacting RNAs.

1. Input and Preprocessing

  • Read input data (FASTQ files) and validate sample information

  • Perform quality control with FASTQC

2. Deduplication (optional)

  • Remove duplicate reads using tools like:

    • fastq-dupaway

    • fastuniq

    • clumpify

3. Trimming

  • Trim low-quality bases and adapters using tools like:

    • fastp

    • Trimmomatic

    • BBduk

    • cutadapt

4. Bridge Processing (for specific experiment types)

  • For experiments like GRID-seq, RADICL-seq, iMARGI, etc., process the bridge sequences that connect RNA and DNA parts

  • Use tools like BBMerge or PEAR to merge paired-end reads

  • Separate RNA and DNA parts based on the bridge sequence

5. Alignment

  • Align RNA and DNA reads to the reference genome using tools like:

    • HISAT2

    • STAR

    • Bowtie2

6. Post-alignment Processing

  • Filter aligned reads for uniqueness and mismatches

  • Convert BAM files to BED format

7. Contact Generation

  • Join RNA and DNA parts to create raw contacts

  • Perform strand detection and correction

8. CIGAR Filtering (optional)

  • Filter contacts based on CIGAR strings to improve quality

9. Merging Replicates

  • Combine data from replicate experiments

10. Chromosome Splitting (optional)

  • Split data by chromosomes for parallel processing

11. Annotation and Voting

  • Annotate RNA parts of contacts using reference annotation

  • Perform voting to resolve conflicting annotations

12. Background Model Generation

  • Create a background model for normalization

13. Normalization

  • Normalize raw contacts using the background model

  • Perform additional normalization steps (N2, scaling)

14. Peak Calling (for One-to-All experiments)

  • Use MACS2 to call significant peaks of chromatin-interacting RNAs

15. Statistics and Visualization

  • Generate statistics at various stages of the pipeline

  • Create plots and visualizations of the results

16. MultiQC Report

  • Compile a comprehensive quality control report using MultiQC

Main Results

The main results of this pipeline are:

  1. Pairs of RNA and DNA contacts, stored in tab-separated files

  2. Annotation of the RNA parts of the contacts

  3. Significant peaks of chromatin-interacting RNAs (for One-to-All experiments)

  4. Various statistics and quality control metrics throughout the process

Note: This pipeline is flexible and can handle different types of all-to-all interactome sequencing data, with options to customize the workflow based on the specific experiment type and analysis requirements.