bio-workflows-chipseq-pipeline

Orchestrates the end-to-end ChIP-seq pipeline from FASTQ to blacklist-filtered, annotated peaks, chaining fastp QC, Bowtie2 alignment, pre-dedup library-complexity QC (NRF/PBC), duplicate removal, chrM + ENCODE-blacklist filtering, MACS3 peak calling against a matched input, IDR/consensus reproducibility, deepTools signal tracks, and ChIPseeker annotation. Use when committing the reference build + blacklist version + effective genome size once, pairing each IP with its matched control, computing complexity metrics BEFORE dedup, choosing narrow vs broad and MACS3 vs SEACR/Genrich, keeping per-replicate peaks for IDR, or avoiding depth-normalization that erases a spike-in global shift. Hands mechanism to the chip-seq component skills; not a re-teach of any single step.

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仓库: GPTomics/bioSkills
最近来源活动: 2026年7月11日 14:11
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SKILL.md

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clawhub-installer

Installs 425 bioinformatics skills covering sequence analysis, RNA-seq, single-cell, variant calling, metagenomics, structural biology, and 56 more categories. Use when setting up bioinformatics capabilities or when a bioinformatics task requires specialized skills not yet installed.

2026-08-11

bio-seq-objects

Create and manipulate Seq, MutableSeq, and SeqRecord objects using Biopython. Use when creating sequences from strings, modifying sequence data in-place, building annotated records for file output, or debugging post-1.78 Bio.Alphabet and immutability errors.

2026-08-11

bio-transcription-translation

Transcribe DNA to RNA and translate to protein using Biopython, with NCBI codon-table selection, CDS validation, and six-frame ORF finding. Use when converting a CDS or ORF to its amino-acid sequence, selecting a non-standard (mitochondrial, bacterial, ciliate) genetic code, validating a coding sequence, or scanning all reading frames.

2026-08-11

查看这个仓库的全部 Skills

ChIP-seq Pipeline

"Process my ChIP-seq data from FASTQ to annotated peaks" -> Chain QC/trim, alignment, pre-dedup complexity QC, dedup + blacklist filtering, control-matched peak calling, reproducibility, signal tracks, and annotation.

CLI + R: fastp -> bowtie2 -> (NRF/PBC pre-dedup) -> samtools markdup -> chrM/blacklist filter -> macs3 callpeak (IP vs input) -> IDR -> bamCoverage -> ChIPseeker

This is a workflow skill: it owns the chaining decisions and hand-offs, not the internals of any one step. Every step below cross-references the component skill that teaches its mechanism.

The governing principle

A ChIP-seq peakset is decided at four seams, not inside the caller.

The reference build + blacklist version + effective genome size is one coordinate commitment made once and inherited by everything downstream — peak coordinates, signal-track scaling, and every overlap. Blacklist filtering is a committed pipeline step, not optional cleanup: ENCODE-blacklisted regions (satellite/rDNA/high-signal artifacts) produce reproducible false peaks in every dataset regardless of biology, so they are removed before calling (Amemiya 2019).

An IP is only interpretable against its matched control — the control IS the enrichment background. Calling peaks without the right input/IgG fabricates peaks at open/accessible and copy-number-amplified regions. Pair each IP with its control at the calling step.

Library-complexity QC (NRF/PBC1/PBC2) is computed on the PRE-dedup BAM. After markdup -r the duplicates are gone, so computing complexity afterward reads ~1.0 and is meaningless. Compute it on the filtered, position-sorted BAM before removing duplicates.

ChIP-seq Pipeline

CLI + R: fastp -> bowtie2 -> (NRF/PBC pre-dedup) -> samtools markdup -> chrM/blacklist filter -> macs3 callpeak (IP vs input) -> IDR -> bamCoverage -> ChIPseeker

This is a workflow skill: it owns the chaining decisions and hand-offs, not the internals of any one step. Every step below cross-references the component skill that teaches its mechanism.

The governing principle

A ChIP-seq peakset is decided at four seams, not inside the caller.

name	bio-workflows-chipseq-pipeline
description	Orchestrates the end-to-end ChIP-seq pipeline from FASTQ to blacklist-filtered, annotated peaks, chaining fastp QC, Bowtie2 alignment, pre-dedup library-complexity QC (NRF/PBC), duplicate removal, chrM + ENCODE-blacklist filtering, MACS3 peak calling against a matched input, IDR/consensus reproducibility, deepTools signal tracks, and ChIPseeker annotation. Use when committing the reference build + blacklist version + effective genome size once, pairing each IP with its matched control, computing complexity metrics BEFORE dedup, choosing narrow vs broad and MACS3 vs SEACR/Genrich, keeping per-replicate peaks for IDR, or avoiding depth-normalization that erases a spike-in global shift. Hands mechanism to the chip-seq component skills; not a re-teach of any single step.
tool_type	mixed
primary_tool	MACS3
workflow	true
depends_on	["read-qc/fastp-workflow","read-alignment/bowtie2-alignment","alignment-files/duplicate-handling","chip-seq/chipseq-qc","chip-seq/peak-calling","chip-seq/peak-annotation","chip-seq/differential-binding","chip-seq/chipseq-visualization","chip-seq/motif-analysis"]
qc_checkpoints	[{"after_qc":"Q30 >85%, adapter content <5%"},{"after_alignment":"Mapping rate >80%, unique mapping >70%"},{"before_dedup":"NRF >0.8, PBC1 >0.8 (computed on the PRE-dedup BAM; after dedup the metric is meaningless)"},{"after_peaks":"FRiP >1% (TF) or >5% (sharp histone; broad marks run lower); NSC >1.05; RSC >0.8; fingerprint separates IP from input"},{"after_idr":"IDR rescue ratio max(Np,Nt)/min and self-consistency ratio max(N1,N2)/min both <=2 (ENCODE); IDR run on PER-REPLICATE peaks"}]

name	bio-workflows-chipseq-pipeline
description	Orchestrates the end-to-end ChIP-seq pipeline from FASTQ to blacklist-filtered, annotated peaks, chaining fastp QC, Bowtie2 alignment, pre-dedup library-complexity QC (NRF/PBC), duplicate removal, chrM + ENCODE-blacklist filtering, MACS3 peak calling against a matched input, IDR/consensus reproducibility, deepTools signal tracks, and ChIPseeker annotation. Use when committing the reference build + blacklist version + effective genome size once, pairing each IP with its matched control, computing complexity metrics BEFORE dedup, choosing narrow vs broad and MACS3 vs SEACR/Genrich, keeping per-replicate peaks for IDR, or avoiding depth-normalization that erases a spike-in global shift. Hands mechanism to the chip-seq component skills; not a re-teach of any single step.
tool_type	mixed
primary_tool	MACS3
workflow	true
depends_on	["read-qc/fastp-workflow","read-alignment/bowtie2-alignment","alignment-files/duplicate-handling","chip-seq/chipseq-qc","chip-seq/peak-calling","chip-seq/peak-annotation","chip-seq/differential-binding","chip-seq/chipseq-visualization","chip-seq/motif-analysis"]
qc_checkpoints	[{"after_qc":"Q30 >85%, adapter content <5%"},{"after_alignment":"Mapping rate >80%, unique mapping >70%"},{"before_dedup":"NRF >0.8, PBC1 >0.8 (computed on the PRE-dedup BAM; after dedup the metric is meaningless)"},{"after_peaks":"FRiP >1% (TF) or >5% (sharp histone; broad marks run lower); NSC >1.05; RSC >0.8; fingerprint separates IP from input"},{"after_idr":"IDR rescue ratio max(Np,Nt)/min and self-consistency ratio max(N1,N2)/min both <=2 (ENCODE); IDR run on PER-REPLICATE peaks"}]

Commitment	Choice	Consequence inherited downstream
Build + blacklist + effective genome size	One genome build; the matching ENCODE blacklist BED; `-g hs/mm`/numeric	Mixed builds mis-place peaks; skipping the blacklist plants reproducible false peaks; wrong `-g` mis-scales p-values
Control pairing	Each IP has its input/IgG	No control => peaks at open chromatin / CN-amplified loci
Peak shape	Narrow (TF, H3K4me3, H3K27ac) vs broad (H3K27me3, H3K36me3, H3K9me3)	Broad marks called with narrow settings fragment into many small peaks
Fragment model	PE: `-f BAMPE` (real fragments); SE: `-f BAM` + `--nomodel --extsize` from predictd/xcorr	BAMPE silently ignores `--shift/--extsize`

Fork	Lean toward	Hand off to
Caller	MACS3 (standard IP+input); SEACR (CUT&RUN/CUT&Tag, low background); Genrich (some ChIP/ATAC, built-in blacklist/replicate handling)	chip-seq/peak-calling, chip-seq/cut-and-run-tag
Narrow vs broad	Narrow: TFs, H3K4me3, H3K27ac. Broad (`--broad --broad-cutoff 0.1`): H3K27me3, H3K36me3, H3K9me3	chip-seq/peak-calling
Reproducibility	ENCODE IDR (per-rep + pooled pseudo-reps) for TFs; naive overlap acceptable for exploratory histone	chip-seq/peak-calling
Consensus set	Pool for a union/consensus set AFTER IDR selects the reproducible threshold	chip-seq/differential-binding

After	Gate	Interpretation
QC/trim	Q30 >85%, adapter <5%	DNA higher quality than RNA
Alignment	Mapping >80%, unique >70%	Low unique = repeats/contamination/wrong build
PRE-dedup	NRF >0.8, PBC1 >0.8	Low complexity = over-amplification/low input; MUST be computed before dedup
Peaks	FRiP >1% (TF) / >5% (sharp histone; broad marks run lower); NSC >1.05; RSC >0.8; fingerprint separates IP/input	Low FRiP/flat fingerprint = weak antibody or failed enrichment (chip-seq/chipseq-qc)
IDR	rescue ratio and self-consistency ratio both <=2	Poor replicate consistency; run IDR on PER-replicate peaks

bio-workflows-chipseq-pipeline

安装方式

检查来源文件

Version Compatibility

ChIP-seq Pipeline

The governing principle

Version Compatibility

ChIP-seq Pipeline

The governing principle

Pipeline map

Made-once commitments

The canonical order and why

Choosing the caller and peak shape

Primary path: Bowtie2 + MACS3 + ChIPseeker

Signal tracks and annotation

QC checkpoints between steps

Common Errors

Pipeline map (hand-offs)

Related Skills

References

Symptom	Cause	Fix
Reproducible peaks over satellite/rDNA/high-signal regions	ENCODE blacklist never subtracted	`bedtools intersect -v` the blacklist BED before calling (committed step)
NRF/PBC ~1.0 and uninformative	Computed after `markdup -r`	Compute complexity on the PRE-dedup, filtered BAM
Peaks at open chromatin / CN-amplified loci	Called without a matched control	Pair each IP with its input/IgG in `callpeak -c`
H3K27me3/H3K9me3 fragmented into many tiny peaks	Broad mark called with narrow settings	Add `--broad --broad-cutoff 0.1`
`--shift/--extsize` had no effect	Used with `-f BAMPE` (ignored for PE)	Use `-f BAM` + `--nomodel` for SE; BAMPE derives fragments
Spike-in global shift disappears in tracks	bamCoverage RPKM/CPM re-equalized depth	`--scaleFactor` + `--normalizeUsing None` (chip-seq/spike-in-normalization)
"IDR" numbers look too good	IDR run on a pooled peakset	Run IDR on per-replicate peaks + pooled pseudo-replicates

bio-workflows-chipseq-pipeline

来源信息

安装方式

检查来源文件

同仓库更多 Skills

同仓库更多 Skills

Version Compatibility

ChIP-seq Pipeline

The governing principle

Version Compatibility

ChIP-seq Pipeline

The governing principle

Pipeline map

Made-once commitments

The canonical order and why

Choosing the caller and peak shape

Primary path: Bowtie2 + MACS3 + ChIPseeker

Signal tracks and annotation

QC checkpoints between steps

Common Errors

Pipeline map (hand-offs)

Related Skills

References