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.