RRBS_pipeline

A lightweight and user-friendly Bash pipeline for Reduced Representation Bisulfite Sequencing (RRBS) data analysis. It performs quality control, adapter trimming, alignment, deduplication, methylation extraction, and summary reporting — all in a single script with optional dry-run mode.

Features

• Supports both single-end and paired-end FASTQ files
• Automatic detection of mode-based arguments
• Creates a detailed log file ({sampleX}.log) with timestamps and command & execution details
• --dry-run option to preview commands without execution
• Clean and minimal dependencies — no workflow engine needed

Requirements

Please ensure the following tools are installed and accessible from your system PATH:

Trim_galore
Bismark
MultiQC
Bowtie2

Usage

chmod +x run_rrbs.sh
./run_rrbs.sh sample_R1.fastq.gz sample_R2.fastq.gz /path/to/bowtie2_index

For single-end:

./run_rrbs.sh sample.fastq.gz /path/to/bowtie2_index

Dry-run mode (preview commands only):

./run_rrbs.sh sample.fastq.gz /path/to/bowtie2_index --dry-run

Output

• *.log — per-sample execution log with steps and timestamps
• *_dedup.bam — deduplicated aligned BAM file
• *_Methylation_report.txt, *.bedGraph.gz, *.cov.gz — methylation calls
• multiqc_report.html — combined quality control summary

Logging

Each major processing step is logged to a file with command and execution details (sampleX.log).

A test example:

RRBS pipeline started at 2025-06-04 07:46:55
Sample: SRR17518177 | Mode: paired-end | Dry-run: true
==========================================

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                                       STAGE 1: FastQC
----------------------------------------------------------------------------------------------------
>> fastqc SRR17518177.R1.fq.gz SRR17518177.R2.fq.gz

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                                       STAGE 2: Trim Galore
----------------------------------------------------------------------------------------------------
>> trim_galore --paired --rrbs SRR17518177.R1.fq.gz SRR17518177.R2.fq.gz

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                                       STAGE 3: Bismark Alignment
----------------------------------------------------------------------------------------------------
>> bismark --genome ~/genomes/hg38/bowtie2_index -1 SRR17518177.R1_val_1.fq.gz -2 SRR17518177.R2_val_2.fq.gz -o .

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                                       STAGE 4: Deduplicate BAM
----------------------------------------------------------------------------------------------------
>> deduplicate_bismark --paired SRR17518177.R1_val_1_bismark_bt2_pe.bam

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                                       STAGE 5: Methylation Extraction
----------------------------------------------------------------------------------------------------
>> bismark_methylation_extractor --paired-end --bedGraph --gzip --no_overlap --output . SRR17518177.R1_val_1_bismark_bt2_pe.deduplicated.bam

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                                       STAGE 6: MultiQC
----------------------------------------------------------------------------------------------------
>> multiqc . --outdir .

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                                       STAGE 7: Cleanup Intermediate Files
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>> rm -f *deduplicated.txt.gz *bt2.bam *M-bias.txt *val_*.fq.gz
>> mv SRR17518177.R1_val_1_bismark_bt2_pe.deduplicated.bam SRR17518177.deduplicated.bam

Pipeline completed at 2025-06-04 07:46:55

========== SUMMARY ==========
Sample name     : SRR17518177
Mode            : paired
Genome index    : ~/genomes/hg38/bowtie2_index
Final BAM       : SRR17518177.deduplicated.bam
Methylation     : SRR17518177.deduplicated_Methylation_report.txt (or .gz/bedGraph)
QC Report       : multiqc_report.html
Log saved to    : SRR17518177.log

License

MIT License

Please let me know if you have any questions or suggestions about my pipeline script!