Dna Methylation Alignment Bismark

DNA Methylation Sequence Alignment with Bismark

Overview

This skill performs bisulfite-aware sequence alignment for DNA methylation sequencing using Bismark only. It is designed for autonomous execution from FASTQ input through aligned BAM generation and basic QC, while preventing unsafe assumptions about genome build, library layout, or assay design.

Main steps include:

• Refer to the Inputs & Outputs section and create the output architecture in Step 0.
• Always ask the user for the reference genome directory to use for Bismark. Never infer genome build from filenames alone.
• Always ask the user whether the assay is WGBS, RRBS, or another bisulfite-based methylation assay if that affects trimming or downstream interpretation and is not already known.
• Detect whether input data are paired-end or single-end.
• Group FASTQ files into samples using naming conventions.
• Validate that the Bismark genome folder has been prepared.
• Run Bismark alignment.
• Sort and index BAM output with samtools.
• Generate basic alignment QC reports and write a parameter log file for every sample.
• Keep the workflow alignment-focused; methylation extraction belongs in a downstream skill.

Inputs & Outputs

Inputs

Accepted FASTQ naming patterns include:

${sample}.fastq.gz
${sample}.fq.gz
${sample}_R1.fastq.gz
${sample}_R2.fastq.gz
${sample}_1.fastq.gz
${sample}_2.fastq.gz

Reference resources must be supplied by the user as one of the following:

/path/to/reference_genome_folder
/path/to/reference.fa

If the user supplies a FASTA, prepare a Bismark genome folder before alignment.

Outputs

all_methylation_alignment/
  aligned_bam/
    ${sample}.sorted.bam
    ${sample}.sorted.bam.bai
  qc/
    ${sample}.flagstat.txt
    ${sample}.idxstats.txt
  logs/
    ${sample}_alignment.log
    ${sample}_used_parameters.txt
  temp/

All outputs must be placed under ${proj_dir} returned in Step 0.

Required User Inputs

The agent must ask for the following when missing:

• Reference genome build
• Bismark genome folder or a reference FASTA to prepare one
• Assay type when biologically relevant and not already known:
  • WGBS
  • RRBS
  • other bisulfite-based methylation assay
• Number of threads if the user has a compute preference; otherwise use a reproducible default
• Whether to keep intermediate BAM files; default is no

The agent must not guess:

• genome build
• assay type when ambiguous
• single-end vs paired-end when file pairing is incomplete or inconsistent
• whether a genome folder prepared for another build/version is acceptable

Decision Logic

Assay Type

Use file and sample names only for tentative classification:

• Names containing WGBS → likely whole-genome bisulfite sequencing
• Names containing RRBS → likely reduced representation bisulfite sequencing
• Names containing only generic terms such as methylation, BSseq, bisulfite → ambiguous

If naming is ambiguous, ask the user.

Library Layout

Use FASTQ grouping rules:

• If files appear as ${sample}_R1 and ${sample}_R2 or ${sample}_1 and ${sample}_2 → paired-end
• If only one FASTQ exists for a sample → single-end
• If an R1 file exists without its mate → stop and ask the user to resolve missing mates

Threads

• Default to 8 threads unless the user specifies otherwise

Step-by-Step Workflow

Step 0: Initialize Project

Create a task directory for methylation alignment outputs.

Suggested call:

• mcp__project-init-tools__project_init

with:

• sample: all
• task: methylation_alignment
• genome: provided by user

The tool will return ${proj_dir}. Use it for all output placement.

If a project-init MCP tool is not available in the runtime, create this directory structure manually:

all_methylation_alignment/
  aligned_bam/
  logs/
  temp/

Set ${proj_dir} to all_methylation_alignment.

Step 1: Detect and Group FASTQ Files

Call:

• mcp__bismark-tools__detect_fastq_samples

with:

• input_dir: directory containing FASTQ files

The tool will:

• find supported FASTQ files
• group them into samples
• detect single-end vs paired-end layout
• report any missing mate files
• return a machine-readable sample summary

Rules:

• Prefer compressed FASTQ (*.fastq.gz, *.fq.gz)
• Use consistent sample grouping
• Fail if file naming is inconsistent or ambiguous

Step 2: Prepare or Validate the Bismark Genome Folder

If the user supplied a FASTA and wants the agent to prepare a Bismark genome folder, call:

• mcp__bismark-tools__prepare_bismark_genome

with:

• reference_fasta: user-provided FASTA path
• genome_folder: destination directory for Bismark genome preparation

If the user supplied an existing Bismark genome folder, validate it before alignment by calling:

• mcp__bismark-tools__validate_bismark_genome

with:

• genome_folder: user-provided genome folder

Stop and ask the user to correct the path if validation fails.

Step 3: Align Each Sample with Bismark

For each detected sample, call:

• mcp__bismark-tools__run_bismark_alignment

with:

• sample_name: sample identifier
• fastq_r1: path to R1 FASTQ or single-end FASTQ
• fastq_r2: path to R2 FASTQ for paired-end data, otherwise omit
• genome_folder: validated Bismark genome folder
• out_dir: ${proj_dir}/aligned_bam
• log_dir: ${proj_dir}/logs
• temp_dir: ${proj_dir}/temp
• threads: user-specified or default 8
• keep_intermediate_bam: false by default

Tool behavior:

• run Bismark only
• write an alignment log
• locate the Bismark BAM output
• sort BAM
• index BAM
• remove intermediate unsorted BAM unless keep_intermediate_bam=true

Expected output:

${proj_dir}/aligned_bam/${sample}.sorted.bam
${proj_dir}/aligned_bam/${sample}.sorted.bam.bai
${proj_dir}/logs/${sample}_alignment.log

This skill is alignment-only. Methylation extraction, deduplication policy, and cytosine report generation belong to downstream skills unless explicitly requested elsewhere.

Step 4: Write Parameter Log

For each sample, the agent must write:

${proj_dir}/logs/${sample}_used_parameters.txt

Example content:

Sample: WGBS_rep1
Assay type: WGBS
Library layout: paired-end
Aligner: Bismark
Reference genome build: hg38
Bismark genome folder: /refs/hg38/bismark_genome
Threads: 8
Intermediate BAM kept: no

Reasoning:
- Sample name contains WGBS, so assay classified as whole-genome bisulfite sequencing
- Paired FASTQ mates were detected automatically
- User provided hg38 Bismark genome folder
- Alignment-only workflow selected; methylation extraction deferred to downstream analysis

Failure Handling

Stop execution and ask the user for correction if any of the following occurs:

• no FASTQ files found
• inconsistent file naming prevents sample grouping
• paired-end mate missing
• Bismark genome folder missing or not prepared
• requested executable not found in PATH
• Bismark returns non-zero exit status
• sorted BAM or BAM index is not created

Do not continue to downstream QC if alignment fails.

Exact MCP Tool Calls Required

1. mcp__bismark-tools__detect_fastq_samples
2. mcp__bismark-tools__prepare_bismark_genome (only when user provides FASTA)
3. mcp__bismark-tools__validate_bismark_genome
4. mcp__bismark-tools__run_bismark_alignment

When the Agent Must Ask the User

The agent must ask before execution when any of the following are missing or ambiguous:

• genome build
• Bismark genome folder or reference FASTA
• assay type when not inferable from context
• whether incomplete FASTQ pairs should be excluded or fixed
• thread count if a project-specific compute policy exists

The agent must not invent these values.