EmbryoLens

Control-FREEC based CNV calling pipeline for preimplantation genetic testing (PGT-A) and embryo aneuploidy detection using a panel-of-normals (PoN) approach.

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Overview

EmbryoLens implements an end-to-end bioinformatics pipeline for aneuploidy and copy number variation (CNV) detection from NGS data of trophectoderm (TE) biopsies. The pipeline uses Control-FREEC for copy number profiling with a custom panel-of-normals (PoN) reference built from euploid embryo samples to improve specificity and reduce noise from low-input, amplified DNA.

Key features:

• Panel-of-normals reference construction and management
• Batch-mode Control-FREEC execution for high-throughput PGT-A
• Autosome copy number uniformity verification from BAM files
• Automated PDF report generation per sample
• Chromosome-level CNV visualization plots

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Repository Structure

EmbryoLens/
├── PoN_new/                      # Panel-of-normals sample directory
├── freec_PoN_m/                  # Control-FREEC PoN reference files
├── scripts/                      # Helper and utility scripts
├── src/                          # Source scripts
├── data/                         # Sample data (excluded from VCS)
├── tmp_pofn/                     # Temporary PoN working files
├── tmp_pofn_bed/                 # Temporary BED format PoN files
├── 562_panel_of_normals.bed      # Pre-built PoN BED reference (n=562)
├── autosome_cnv_check.sh         # Autosome copy number verification QC
├── chr_freec_graph_batch_*.sh    # Batch chromosome CNV graph generation
├── freec_bed_pon_batch.sh        # Batch FREEC run with BED-based PoN
├── freec_pon_batch.sh            # Batch FREEC run with PoN reference
├── freec_results_pdf.py          # PDF report generator (v1)
├── freec_results_pdf_v2.py       # PDF report generator (v2, improved)
├── freec_results_pdf_v3.py       # PDF report generator (v3, with QC metrics)
├── freec_results_pdf_v4.py       # PDF report generator (v4, final production)
└── make_chr_freec_graph.sh       # Per-sample chromosome-level CNV graph

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Pipeline Workflow

BAM Files (TE biopsy, WGA amplified)
    │
    ▼
1. Autosome CNV Verification      (autosome_cnv_check.sh)
   └─ Per-chromosome (chr1-chr22) read depth profiling
   └─ Coefficient of variation (CV) QC flag: PASS / WARN
    │
    ▼
2. Control-FREEC CNV Calling      (freec_pon_batch.sh)
   └─ Runs FREEC against PoN reference (n=562)
   └─ Computes copy number and BAF profiles
    │
    ▼
3. Chromosome Graph               (chr_freec_graph_batch.sh)
   └─ Per-chromosome CNV visualization
    │
    ▼
4. Report Generation              (freec_results_pdf_v4.py)
   └─ Clinical PDF report with CNV calls per sample
    │
    ▼
PGT-A Result Report (.pdf)

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Requirements

Tool	Version	Purpose
Control-FREEC	≥ 11.6	CNV calling engine
SAMtools	≥ 1.15	BAM processing and autosome CNV QC
Python	≥ 3.8	PDF report generation
R	≥ 4.0	CNV graph generation
Bash	≥ 4.0	Pipeline orchestration

Python dependencies:

pip install reportlab matplotlib pandas numpy

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Installation

git clone https://github.com/rvrane/EmbryoLens.git
cd EmbryoLens
chmod +x *.sh

Ensure Control-FREEC is installed and available in $PATH:

# Install Control-FREEC
git clone https://github.com/BoevaLab/FREEC.git
cd FREEC/src && make
export PATH=$PATH:/path/to/FREEC/src

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Usage

Step 1: Autosome Copy Number Verification

bash autosome_cnv_check.sh /path/to/sample.bam

This step computes per-chromosome read depth across chr1–chr22 and calculates the coefficient of variation (CV) as a QC metric. Samples with CV > 25% are flagged as WARN and should be reviewed before proceeding.

Step 2: Run batch Control-FREEC with PoN reference

bash freec_pon_batch.sh \
  --bam_dir /path/to/bams/ \
  --pon_ref 562_panel_of_normals.bed \
  --genome hg38 \
  --threads 8

Step 3: Generate chromosome-level CNV graphs

bash chr_freec_graph_batch_20250805.sh --output_dir /path/to/output/

Step 4: Generate PDF reports

python freec_results_pdf_v4.py \
  --freec_dir /path/to/freec_output/ \
  --output_dir /path/to/reports/

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Output

File/Directory	Description
*.bam_CNVs	Raw CNV calls per sample from FREEC
*.bam_ratio.txt	Normalized copy number ratios
chr_plots/	Per-chromosome CNV visualization
reports/*.pdf	Clinical PGT-A result PDFs
autosome_qc.log	Autosome CNV verification QC log

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Panel-of-Normals (PoN)

The included 562_panel_of_normals.bed file was constructed from n=562 euploid control embryos sequenced with a targeted low-pass NGS approach. The PoN is used to normalize sample CNV profiles against background noise from whole genome amplification (WGA) artifacts.

Note: For clinical use, it is recommended to rebuild the PoN using your own lab's euploid controls to match your specific library prep and sequencing platform.

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References

1. Boeva V, et al. Control-FREEC: a tool for assessing copy number and allelic content using next-generation sequencing data. Bioinformatics. 2012;28(3):423-5. PMID: 22155870
2. Boeva V, et al. FREEC: a tool for copy number analysis. Bioinformatics. 2011;27(2):268-9. PMID: 21081509

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Author

Rugved Rane
Senior Bioinformatician | Reproductive Genetics & Clinical Genomics
GitHub

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License

MIT License — see LICENSE for details.