bio-workflows-clip-pipeline

Version Compatibility

Reference examples tested with: FastQC 0.12+, STAR 2.7.11+, bedtools 2.31+, cutadapt 4.4+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

CLIP-seq Pipeline

"Analyze my CLIP-seq data from FASTQ to binding sites and motifs" → Orchestrate UMI extraction, adapter trimming, STAR alignment, PCR deduplication, CLIPper/PureCLIP peak calling, binding site annotation, and HOMER motif enrichment.

Pipeline Overview

FASTQ → QC → UMI extract → Trim adapters → Align → Filter → Dedup → Peak call → Annotate → Motifs

CLIP Method Variants

Method	UMI	Crosslink Site	Adapter
HITS-CLIP	Optional	Deletions	3' adapter
PAR-CLIP	Optional	T→C mutations	3' adapter
iCLIP	Required	5' of read	3' adapter
eCLIP	Required	5' of read	3' adapter

Step 1: Quality Control

# Initial QC
fastqc reads.fastq.gz -o qc_pre/

# Check for adapter contamination and UMI structure
# For eCLIP: expect 10nt UMI at read start
zcat reads.fastq.gz | head -n 100 | cut -c1-15

Step 2: UMI Extraction

# eCLIP (10nt UMI at 5' end)
umi_tools extract \
    --stdin=reads.fastq.gz \
    --bc-pattern=NNNNNNNNNN \
    --stdout=extracted.fastq.gz \
    --log=umi_extract.log

# iCLIP (5nt experimental barcode + 5nt UMI)
umi_tools extract \
    --stdin=reads.fastq.gz \
    --bc-pattern=NNNNNXXXXX \
    --stdout=extracted.fastq.gz

Step 3: Adapter Trimming

# Trim 3' adapter (common eCLIP adapter)
cutadapt -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCA \
    --minimum-length 20 \
    --quality-cutoff 20 \
    -o trimmed.fastq.gz \
    extracted.fastq.gz

# For paired UMI adapters
cutadapt -a AGATCGGAAGAGCACACGTCT \
    -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT \
    --minimum-length 20 \
    -o trimmed_R1.fq.gz -p trimmed_R2.fq.gz \
    extracted_R1.fq.gz extracted_R2.fq.gz