Improve Confidence in Exon-Level BRCA1 & BRCA2 CNV Detection
Pathogenic variants in BRCA1 and BRCA2 are among the most extensively studied genetic alterations associated with hereditary breast and ovarian cancer (HBOC). While much attention is given to single nucleotide variants (SNVs) and small insertions/deletions (indels), copy number variants (CNVs)—including exon-level deletions and duplications—represent an important class of disease-causing mutations that can be missed if testing strategies are not optimized for their detection.
Accurate identification of these structural changes requires more than simply generating sequencing data. Reliable CNV analysis depends on highly uniform target coverage, sufficient sequencing depth, and robust normalization methods capable of distinguishing true biological variation from technical noise.
At Paragon Genomics, we developed the CleanPlex® BRCA1 & BRCA2 Kit v3 to generate highly uniform amplicon sequencing libraries that provide the consistency required for high-resolution exon-level CNV analysis.
Our latest technical note demonstrates how read-depth normalization, combined with orthogonal sequencing evidence, can accurately identify clinically relevant BRCA1 exon deletions and duplications using targeted amplicon sequencing.
What are BRCA Copy Number Variants?
BRCA1 and BRCA2 function as tumor suppressor genes responsible for repairing double-stranded DNA breaks through homologous recombination. When these genes lose function through inherited pathogenic variants, DNA repair becomes impaired, leading to genomic instability and an increased lifetime risk of several cancers, including breast, ovarian, pancreatic, and prostate cancers.
Although many pathogenic BRCA mutations are point mutations or small indels, large genomic rearrangements—including exon-level deletions and duplications—can account for a meaningful proportion of pathogenic BRCA variants in hereditary cancer testing. Depending on the population studied and testing cohort, these rearrangements may represent several percent of all pathogenic BRCA findings, making their detection an important component of comprehensive genetic analysis.
Because CNVs alter the number of copies of one or more exons rather than changing individual nucleotide sequences, they require specialized analytical approaches that evaluate sequencing coverage across targeted regions rather than conventional variant calling alone.
Why is BRCA CNV Detection Challenging?
Unlike SNVs, copy number variants cannot be identified simply by examining sequence differences. Instead, CNV detection relies on measuring relative sequencing coverage across targeted regions. Small variations in PCR amplification efficiency, GC content, sequencing depth, primer performance, or sample quality can all introduce fluctuations in read counts that resemble copy number changes.
Without careful normalization, these technical artifacts can obscure true biological events or generate false positives.
Several factors make exon-level CNV detection particularly challenging:
- Differences in amplification efficiency between amplicons
- Variable GC content across BRCA exons
- Sequencing depth differences between samples
- PCR bias introduced during library preparation
- Sample quality and DNA input variability
- Distinguishing true single-exon events from normal technical variation
For these reasons, successful CNV detection depends on both robust laboratory performance and sophisticated bioinformatic normalization.
Why High Uniformity Matters
Coverage uniformity is one of the most important factors affecting read-depth-based CNV detection.
When sequencing coverage varies widely across target regions, distinguishing true copy number changes from normal assay variability becomes increasingly difficult. High uniformity allows each exon to be compared against a stable baseline, improving confidence when identifying heterozygous deletions, duplications, and larger copy number events.
The CleanPlex® chemistry was designed to generate highly uniform amplification across thousands of targeted amplicons while maintaining low DNA input requirements and a streamlined workflow.
This consistency provides a strong foundation for quantitative read-depth analysis and reliable exon-level CNV detection.
The CleanPlex Advantage
Unlike hybrid capture workflows that often require higher DNA input and longer library preparation times, CleanPlex utilizes multiplex PCR to efficiently enrich target regions while maintaining excellent coverage uniformity.
Key advantages include:
- Highly uniform target coverage
- Low DNA input requirements
- Rapid single-tube workflow
- High sequencing depth across target exons
- Cost-effective targeted sequencing
- Robust performance across clinically relevant BRCA regions
These characteristics make CleanPlex well suited for targeted hereditary cancer research workflows where consistent exon coverage is essential for downstream CNV analysis.
A Multi-Layered Approach to CNV Detection
Rather than relying solely on raw sequencing depth, the workflow described in our technical note applies multiple layers of analysis to improve confidence in CNV detection.
The workflow includes:
Intra-sample normalization
Corrects for differences in total sequencing depth within each sample while accounting for characteristics such as amplicon length and primer pool.
Inter-sample normalization
Removes systematic amplicon-specific bias by comparing normalized read counts across the sequencing cohort.
Read-depth ratio analysis
Normalized coverage ratios are evaluated against expected diploid baselines to identify potential exon deletions and duplications.
Orthogonal sequencing evidence
Additional evidence (including loss of heterozygosity (LOH), soft-clipped reads, and gapped read alignments) provides independent support for detected CNV events, increasing confidence in the final interpretation.
Technical Validation Using Coriell Reference Samples
To evaluate the workflow, Paragon Genomics analyzed multiple well-characterized Coriell reference cell lines containing known BRCA1 copy number variants.
The study successfully identified:
- Heterozygous deletion in BRCA1 exon 10
- Heterozygous duplication in BRCA1 exon 12
- Heterozygous deletion spanning BRCA1 exons 14–15
Observed normalized read-depth ratios closely matched the known reference profiles, while IGV visualization demonstrated orthogonal evidence supporting each event through alignment signatures and loss of heterozygosity. Together, these findings demonstrate the feasibility of using highly uniform amplicon sequencing data for reliable exon-level CNV detection.
Download the Technical Note
Learn how Paragon Genomics demonstrated exon-level BRCA CNV detection using targeted amplicon sequencing.
Inside the technical note you’ll find:
- Background on BRCA CNV biology
- Read-depth normalization methodology
- Study design using Coriell reference materials
- Example CNV plots
- IGV visualization of deletions and duplications
- Loss of heterozygosity analysis
- Soft-clipping and gapped alignment evidence
- Complete study conclusions and workflow
Why Researchers Choose CleanPlex®
The CleanPlex® BRCA1 & BRCA2 Kit v3 combines rapid library preparation, exceptional coverage uniformity, and high sequencing depth to support targeted hereditary cancer research.
If your laboratory is developing hereditary cancer assays, validating targeted sequencing workflows, or evaluating methods for exon-level CNV analysis, CleanPlex provides the consistency that is needed for accurate read-depth-based copy number assessment.
Frequently Asked Questions
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The CleanPlex BRCA1 & BRCA2 Kit v3 is a targeted resequencing assay built for BRCA testing, covering all exonic regions and flanking intronic sequences of BRCA1 and BRCA2. It's designed to support labs performing clinical diagnostics research by identifying disease-relevant variants with high confidence, using next-generation sequencing to generate accurate, reproducible data.
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Yes. The multiplex PCR-based workflow enables analysis of both somatic and germline variants in a single assay, with sensitivity down to 1% variant allele frequency using just 20 ng of input DNA. This makes the kit well suited for identifying germline mutations alongside low-frequency somatic changes in the same sample.
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The CleanPlex BRCA1 & BRCA2 Kit v3 has highly uniform amplicon coverage and consistent coverage depth across target exons, making it an effective CNV panel for identifying exon-level deletions and duplications. This uniformity reduces technical noise, supporting reliable detection of clinically relevant copy number changes that could otherwise be missed.
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Starting from 20 ng of DNA, the CleanPlex BRCA1 & BRCA2 Kit v3 uses a streamlined, three-step library preparation protocol that generates sequencing-ready libraries in about 3 hours. This fast turnaround, combined with excellent on-target performance, makes it practical for labs processing a broad range of sample types and volumes.
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The CleanPlex BRCA1 & BRCA2 Kit v3 is specifically designed to target DNA variants within BRCA1 and BRCA2 exons and their flanking regions. It isn't intended for other samples or genes outside this panel. However, Paragon Genomics offers additional CleanPlex panels for other hereditary cancer genes, and the BRCA panel also integrates with VarSome Clinical for streamlined variant annotation and interpretation.
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BRCA1 and BRCA2 mutations are some of the most well-established genetic contributors to hereditary breast cancer; the CleanPlex BRCA1 & BRCA2 Kit v3 targeted design makes it a very practical tool for researchers who are studying inherited cancer risk. Its rapid workflow and low DNA input make it easy to incorporate into broader hereditary cancer research programs.