Alignment Quality Control for ChIP-seq/ATAC-seq

Overview

Perform comprehensive preliminary alignment-level quality control for ChIP-seq and ATAC-seq BAM files using samtools, Picard, and MultiQC.

Main steps include:

• Initialize the project directory.
• Refer to the Inputs & Outputs section to check inputs and build the output architecture. All the output file should located in ${proj_dir} in Step 0.
• Sort and add read groups if missing in the BAM file.
• Run preliminary QC metrics
• Generate MultiQC report

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When to use this skill

• Use skill when you want to perform alignment-level quality control for ChIP-seq or ATAC-seq BAM files.

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Inputs & Outputs

Inputs

<sample>.bam

Outputs

alignment_qc/
  ${sample}.bam                        # Original input
  ${sample}.sorted.bam                 # (Optional) Created if sorting was needed
  ${sample}.RG.bam                     # (Optional) Created if RG was needed
  ${sample}.RG.bam.bai                 # Index file
  qc_results/
    ${sample}.flagstat.txt
    ${sample}.stats.txt
    ${sample}.insertsize_metrics.txt
    ${sample}.dup_metrics.txt
    alignment_qc_report.html      # Visual MultiQC report
    qc_summary.txt                # Pass/Warn/Fail table
  temp/
    ${sample}.markdup.bam            # Intermediate file (safe to delete later)
    ...

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Decision Tree

Step 0: Initialize Project

Call:

• mcp__project-init-tools__project_init

with:

• sample: all
• task: alignment_qc

The tool will:

• Create ${sample}_alignment_qc directory.
• Return the full path of the ${sample}_alignment_qc directory, which will be used as ${proj_dir}.

Step 1: Check and Fix BAMs

• Ensure all BAM files are coordinate-sorted, have Read Groups, and are indexed. This tool will skip files that are already correct and only create temporary files when fixes are needed.

Call:

• mcp__qc-tools__check_and_fix_bams

with:

• bam_files: List of BAM files to process.
• temp_dir: ${proj_dir}/temp

Step 2: Run Alignment QC Metrics

Call:

• mcp__qc-tools__run_bam_qc

with:

• bam_files: List of BAM files to process.
• qc_dir: ${proj_dir}/qc_results
• temp_dir: ${proj_dir}/temp

Step 3: Generate Summary Report

Call:

• mcp__qc-tools__generate_qc_report

with:

• qc_dir: ${proj_dir}/qc_results

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Quality Assessment

Key QC Metrics

• Total reads – overall sequencing depth
• Mapped reads (%) – alignment efficiency
• Properly paired (%) – valid pair fraction (paired-end)
• Duplicate rate (%) – PCR duplication estimate
• Mitochondrial reads (%) – mitochondrial contamination
• Insert size distribution – fragment length profile

All metrics are derived from samtools/Picard and summarized by MultiQC.

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Quality Thresholds

| Category | Criteria | Interpretation | |-----------|-----------|----------------| | Pass | All metrics within recommended thresholds | Suitable for downstream analysis | | Warn | One or more borderline metrics | Likely acceptable; review recommended | | Fail | Critical metrics outside acceptable ranges | Re-sequencing or reprocessing suggested |

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Report Generation

After MultiQC completes, generate a sample-wise summary (PASS/WARN/FAIL) per thresholds in references/qc_metrics.md and save it as:

qc_results/qc_summary.txt

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Resources

Use references/qc_metrics.md for:

• Metric definitions and recommended thresholds
• Troubleshooting guidance
• Readiness criteria for peak calling
• Pointers to ENCODE/nf-core QC standards