Replicates Incorporation Skill

Overview

This skill provides two modes for replicates incorporation:

• 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.
• Always use filtered BAM file (*.filtered.bam) if available.
• Always prompt user for whether generate psedo-replicates if more then 2 replicates.
• Pre-Peak Calling (BAM Mode): If provided with >2 biological replicates, it merges all BAMs, generate the merge BAM file to prepare for track generation and splits them into 2 balanced "pseudo-replicates" to prepare for peak calling only if user required.
• Post-Peak Calling (Peak Mode): If provided with peak files (only support two replicates, derived from either 2 true replicates or 2 pseudo-replicates), it performs IDR (Irreproducible Discovery Rate) analysis, filters non-reproducible peaks, and generates a final "conservative" or "optimal" consensus peak set

Decision Tree

Step 0: Initialize Project

Call:

• mcp__project-init-tools__project_init

with:

• sample: all
• task: rep_merge

The tool will:

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

Pre-Peak Calling (BAM Mode)

Call:

• mcp__bw_tools__pool_bams with:
• bam_files: [${rep1_bam}, ${rep2_bam}, ${rep3_bam}] (Add as many as needed)
• output_bam: ${proj_dir}/temp/${sample}.pooled.bam

Call: (call this only when more than two replicates are provided and user prompt for generating pseudo replicates)

• mcp__bw_tools__split_pseudo_replicates with: bam_file: ${proj_dir}/temp/${sample}.pooled.bam output_rep1: ${proj_dir}/temp/${sample}.pseudo1.bam output_rep2: ${proj_dir}/temp/${sample}.pseudo2.bam

Post-Peak Calling (Peak Mode)

A. Narrow Peaks / ATAC (IDR) Use this to combine reproducible peaks. You should ideally run IDR on:

1. True Replicates
2. Pseudo-Replicates

Call:

• mcp__bw_tools__filter_idr_peaks with:
• peak_file_a: Path to Replicate 1 narrowPeak file.
• peak_file_b: Path to Replicate 2 narrowPeak file.
• output_optimal: ${proj_dir}/peaks/${sample}.idr.narrowPeaks
• output_raw_idr: ${proj_dir}/temp/${sample}_idr_results.tsv
• input_file_type: narrowPeak
• rank_measure: q.value

B. Broad Peaks (Consensus) Call:

• mcp__bw_tools__merge_consensus_peaks with: peak_file_a: Path to Replicate 1 broadPeak file. peak_file_b: Path to Replicate 2 broadPeak file. output_peak: ${proj_dir}/peaks/${sample}.consensus.broadPeaks overlap_fraction: 0.5

Best Practices

• Use pooled tracks for visualization and differential analysis.
• Keep individual replicate tracks for QC and reproducibility evaluation.
• Use IDR ≤ 0.05 for reproducible narrow ChIP-seq peaks and ATAC-seq.
• **Use overlap ≥50% ** for broad histone mark peaks.