Methylation Analysis Pipeline

This Nextflow pipeline performs differential methylation analysis from Bismark or methyldackel output. It identifies differentially methylated regions, annotates them to genes, and performs enrichment analysis.

Input Files

samplesheet - present in the root dir for testing. This will work with both the file types.

genes.gtf s3 path - s3://refdata-1/nfcore-pipelines/bio-data/Homo_sapiens/NCBI/GRCh38/Annotation/Genes/genes.gtf

input-bismark-path - s3://quark-dev-data/quarkdata/home/quark/ayam-invisibl-io/myresults/jobs/methylseq-bismark-b1-tt294/

input-methyldackel-path - s3://quark-dev-data/quarkdata/home/quark/ayam-invisibl-io/myresults/jobs/methylseq-bwa-b1-9vrg9/

Quickstart

1. Build Docker Image: bash docker build -t methylseq .

2. Run the Pipeline: bash nextflow run main.nf --samplesheet <path_to_samplesheet> --input_dir <path_to_input_data> --gtf <path_to_gtf>

Parameters

Mandatory

• --samplesheet: Path to the samplesheet CSV file. The file should contain the following columns:
  • sample: Sample name
  • cohort: Cohort or group for each sample
  • genome: Genome assembly (e.g., hg38)
• --gtf: Path to the gene transfer format (GTF) file for gene annotation.

Optional

• --input_dir: Path to the directory containing methylation data (Bismark coverage or methyldackel output).
• --file_type: Type of input files. Choose from bismark or methyldackel. (Default: bismark)
• --outdir: Output directory for results. (Default: ./results)
• --min_coverage: Minimum read coverage to include a CpG site. (Default: 10)
• --meth_diff_threshold: Absolute methylation difference threshold for calling significant regions. (Default: 25)
• --qval_threshold: Q-value (adjusted p-value) threshold for significance. (Default: 0.05)
• --top_gene_count: Number of top hyper- and hypo-methylated genes to select for enrichment analysis. (Default: 10)
• --enrichr_databases: Comma-separated list of Enrichr databases for enrichment analysis. (Default: See nextflow.config)

Examples

Bismark Analysis

This example runs the pipeline on Bismark output, with a minimum coverage of 10 and a q-value threshold of 0.01.

bash nextflow run main.nf \ --samplesheet samplesheet.csv \ --input_dir bismark/ \ --gtf genes.gtf \ --file_type bismark \ --min_coverage 10 \ --qval_threshold 0.01 \ --outdir results_bismark/

Methyldackel Analysis

This example runs the pipeline on methyldackel output, with a higher minimum coverage and a custom output directory.

bash nextflow run main.nf \ --samplesheet samplesheet.csv \ --input_dir methyldackel/ \ --gtf genes.gtf \ --file_type methyldackel \ --min_coverage 25 \ --outdir results_methyldackel/

Tested With the following command nextflow run main.nf \ --samplesheet methylseq-samplesheet.csv \ --gtf genes.gtf \ --filetype bismark \ --mincoverage 10 \ --qvalthreshold 0.01 \ --outdir resultsbismark/