Overview

This Nextflow pipeline is designed for the analysis of Twist NGS Methylation data, including quality control, alignment, methylation calling, differential methylation analysis, and post-processing. It integrates various tools and custom scripts to provide a comprehensive analysis workflow.

Features

Step	Workflow
Generate Reference Genome Index (optional)	Bismark
Raw data QC	FastQC
Adapter sequence trimming	Trim Galore
Align Reads	Bismark (bowtie2)
Deduplicate Alignments	Bismark
Sort and indexing	Samtools
Extract Methylation Calls	Bismark
Sample Report	Bismark
Summary Report	Bismark
Alignment QC	Qualimap
QC Reporting	MultiQC
Differential Methylation Analysis	EdgeR/MethylKit
Post processing	ggplot2
GO analysis	Gene Ontology

Pipeline Schema

Requirements

• Nextflow (>=21.10.3)
• Docker or Singularity (for containerized execution)
• Java (>=8)

Usage

--diff_meth_method EdgeR

# when using the reference genome indexing, --genome_fasta

nextflow run JD2112/TwistNext \
    -profile singularity \
    --sample_sheet Sample_sheet_twist.csv \
    --genome_fasta /data/reference_genome/hg38/hg38.fa \ 
    --diff_meth_method edger \
    --outdir Results/TwistNext_edgeR 


# if you already have the bisulfite genome index, --bismark_index

nextflow run JD2112/TwistNext \
    -profile singularity \
    --sample_sheet Sample_sheet_twist.csv \
    --bismark_index /data/reference_genome/hg38/ \ 
    --diff_meth_method edger \
    --outdir /mnt/Results/TwistNext_edgeR 

--diff_meth_method MethylKit

# when using the reference genome indexing, --genome_fasta

nextflow run JD2112/TwistNext \
    -profile singularity \
    --sample_sheet Sample_sheet_twist.csv \
    --genome_fasta /data/reference_genome/hg38/hg38.fa \ 
    --diff_meth_method methylkit \
    --outdir Results/TwistNext_methylKit 


# if you already have the bisulfite genome index, --bismark_index

nextflow run JD2112/TwistNext \
    -profile singularity \
    --sample_sheet Sample_sheet_twist.csv \
    --bismark_index /data/reference_genome/hg38/ \ 
    --diff_meth_method methylkit \
    --outdir Results/TwistNext_methylKit 

HELP

nextflow run JD2112/TwistNext --help --outdir .

Find the details on the manual

Credits

• Main Author:

  • Jyotirmoy Das (@JD2112)

• Collaborators:

  • Debojyoti Das (@BioDebojyoti)
  • Leila Nasirzadeh (@Lailanasd)

Citation

Das, J. (2024). TwistNext (v1.0.0). Zenodo. https://doi.org/10.5281/zenodo.14204261

HELP/FAQ/Troubleshooting

Please check the manual for details.

Please create issues on github.

License(s)

GNU-3 public license.

Acknowledgement

We would like to acknowledge the Core Facility, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden and Clinical Genomics Linköping, Science for Life Laboratory, Sweden for their support.