usage
The pipeline needs to use NextFlow, Singularity/Docker/Conda (for container). Details are given in corresponding pages.
It is tested locally on Ubuntu server 24.04.2 LTS.
Tested on HPC (Dardel from PDC, KTH)
Computational Requirements
Software/s Requirements
Docker or Singularity (for containerized execution)
Java (>=8)
Hardware Requirements
RAM: 12 GB - 200 GB
CPU: min. 12 cores
Storage: ~2TB for 24 paired-end samples
Run NextFlow
nextflow run JD2112/TwistMethylFlow \
-profile singularity \
--sample_sheet Sample_sheet_twist.csv \
--genome_fasta /data/reference_genome/hg38/hg38.fa \
--run_both_methods \
--gtf_file /data/Homo_sapiens.GRCh38.104.gtf \
--refseq_file /data/hg38_RefSeq.bed.gz \
--outdir Results/TwistMethylFlow_both
??? info "additional options" Run with pre-build reference genome index --bismark_index /data/reference_genome/hg38/
**Run only EdgeR**
`--diff_meth_method edger`
**Run only MethylKit**
`--diff_meth_method methylkit`
??? warning "need annotation files" Remember to add annotation files for different differential methylation analysis
`--gtf_file /data/Homo_sapiens.GRCh38.104.gtf` for **MethylKit**
`--refseq_file /data/hg38_RefSeq.bed.gz` for **EdgeR**
??? note "Help" nextflow run main.nf --help --outdir .
Parameters configuration
User can change the conf/params.config
or use the --
flags directly on nextflow command line.
--sample_sheet
Path to the sample sheet CSV file (required)
--bismark_index
Path to the Bismark index directory (required unless --genome
or --aligned_bams
is provided)
--genome
Path to the reference genome FASTA file (required if --bismark_index
not provided)
--aligned_bams
Path to aligned BAM files (use this to start from aligned BAM files instead of FASTQ files)
--refseq_file
Path to RefSeq file for annotation (reuired to run both
or methylkit
)
--gtf_file
Path to GTF file for annotation (reuired to run both
or edger
)
--outdir
Output directory (default: ./results)
--diff_meth_method
Differential methylation method to use: 'edger' or 'methylkit' (default: edger)
--run_both_methods
Run both edgeR and methylkit for differential methylation analysis (default: false)
--skip_diff_meth
Skip differential methylation analysis (default: false)
--coverage_threshold
Minimum read coverage to consider a CpG site (default: 10)
--logfc_cutoff
Differential methylation cut-off for Volcano or MA plot (default: 1.5)
--pvalue_cutoff
Differential methylation P-value cut-off for Volcano or MA plot (default: 0.05)
--hyper_color
Hypermethylation color for Volcano or MA plot (default: red)
--hypo_cutoff
Hypomethylation color for Volcano or MA plot (default: blue)
--nonsig_color
Non-significant color for Volcano or MA plot (default: black)
--compare_str
Comparison string for differential analysis (e.g. "Group1-Group2")
--top_n_genes
Number of top differentially methylated genes to report for GOplot (default: 100)
--help
Show this help message and exit
-r
Run with --tag
version from GitHub (e.g, -r 1.0.5
)
??? info "MethylKit specific parameters" --assembly
- user needs to provide the genome assembly version. Default: hg38
`--mc_cores` - if required to run on multiple cores. Default: `1`
`--diff` - Differential methylation cutoff value. Default: `0.5`
`--qvalue` - qvalue respect to differential methylation value to identify significant CpGs. Default: `0.05`
??? note "Other parameters configuration" 1. conf/resource.config
- for resource settings. 2. conf/base.config
- for base settings. 3. nextflow.config
- for nextflow run with default setting. 4. dag.config
- for DAG configuration settings.
Input File
Sample sheet (CSV format) with sample information
Sample_sheet.csv
:
12A
VD
FASTQ/12A_S9_L001_R1_001.fastq.gz
FASTQ/12A_S9_L001_R2_001.fastq.gz
13A
CS
FASTQ/13A_S11_L001_R1_001.fastq.gz
FASTQ/13A_S11_L001_R2_001.fastq.gz
1A
CS
FASTQ/1A_S1_L001_R1_001.fastq.gz
FASTQ/1A_S1_L001_R2_001.fastq.gz
20A
VD
FASTQ/20A_S13_L001_R1_001.fastq.gz
FASTQ/20A_S13_L001_R2_001.fastq.gz
21A
VD
FASTQ/21A_S15_L001_R1_001.fastq.gz
FASTQ/21A_S15_L001_R2_001.fastq.gz
22A
VD
FASTQ/22A_S17_L001_R1_001.fastq.gz
FASTQ/22A_S17_L001_R2_001.fastq.gz
23A
VD
FASTQ/23A_S19_L001_R1_001.fastq.gz
FASTQ/23A_S19_L001_R2_001.fastq.gz
25A
CS
FASTQ/25A_S21_L001_R1_001.fastq.gz
FASTQ/25A_S21_L001_R2_001.fastq.gz
26A
VD
FASTQ/26A_S23_L001_R1_001.fastq.gz
FASTQ/26A_S23_L001_R2_001.fastq.gz
2A
CS
FASTQ/2A_S3_L001_R1_001.fastq.gz
FASTQ/2A_S3_L001_R2_001.fastq.gz
3A
VD
FASTQ/3A_S5_L001_R1_001.fastq.gz
FASTQ/3A_S5_L001_R2_001.fastq.gz
5A
CS
FASTQ/5A_S7_L001_R1_001.fastq.gz
FASTQ/5A_S7_L001_R2_001.fastq.gz
Results
Here is how the result folder looks like -
.
├── annotate_results
├── bismark_align
├── bismark_deduplicate
├── bismark_genome_preparation
├── bismark_methylation_extractor
├── bismark_report
├── edger_analysis
├── fastqc
├── go_analysis
├── multiqc
├── pipeline_info
├── post_processing
├── qualimap
├── samtools_index
├── samtools_sort
└── trim_galore
??? info "bismark alignment with bowtie2" | Option | Functionality | | ---------------------- | --------------------------------------------------------------------------- | | -q
| Quiet mode: suppresses detailed output. | | --score-min L,0,-0.2
| Sets a linear minimum score for valid alignments (moderate stringency). | | --ignore-quals
| Ignores base quality scores during alignment. | | --no-mixed
| Ensures both ends of paired reads align properly; no single-end alignments. | | --no-discordant
| Prevents discordant alignments; enforces proper orientation and distance. | | --dovetail
| Allows overlapping or extended alignments in paired-end reads. | | --maxins 500
| Sets the maximum allowed distance between paired-end reads to 500 bases. |
??? tip "SLURM script to run the sample data on HPC cluster" Here is an example SLURM script to run the pipeline on a HPC cluster with Singularity:
```bash
#!/bin/bash
#SBATCH --job-name=TMF_test
#SBATCH --partition=standard
#SBATCH --nodes=2
#SBATCH --mem=96G
#SBATCH --time=3-00:00:00 # 3 days (D-HH:MM:SS)
#SBATCH --cpus-per-task=16
#SBATCH --output=tmf_test%j.out
#SBATCH --error=tmf_test%j.err
#SBATCH --mail-user=jyotirmoy.das@liu.se
#SBATCH --mail-type=BEGIN,END,FAIL
set -e
# Load modules
module load singularity-4.1.1
module load nextflow-25.04.0
# Set Nextflow and Singularity directories
export NXF_HOME=<Your Nextflow home directory, e.g., /home/username/.nextflow >
export NXF_WORK=<Your Nextflow home directory, e.g., /home/username/nextflow_work>
export SINGULARITY_CACHEDIR=<Your Singularity cache directory, e.g., /home/username/singularity_cache>
# Ensure directories exist
mkdir -p $NXF_WORK $SINGULARITY_CACHEDIR
# Run Nextflow
nextflow run JD2112/TwistMethylFlow \
-profile singularity \
--sample_sheet samplesheet.csv \
--genome_fasta <ABSOLUTE\ PATH\ TO\>/reference_genome/hg19.fa \
--run_both_methods \
--refseq_file hg19_RefSeq.bed.gz \
--gtf_file Homo_sapiens.GRCh37.75.formatted.gtf \
--outdir TMF_250519 \
-with-dag
```
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