BRCA1/2 NGS Assay for Exon Copy Number Variation Detection
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Solution Overview
Problem
Traditional sequencing approaches find it difficult to detect large rearrangements in BRCA1 and BRCA2 genes, such as exon-level copy number variations, which are important for cancer diagnostics, especially in formalin-fixed paraffin-embedded (FFPE) samples.
Innovation Solution
A next-generation sequencing (NGS) assay using a kit with primers targeting BRCA1 and BRCA2 genes, amplifying nucleic acid samples to produce overlapping amplicons, sequencing these amplicons, mapping reads to a reference sequence, determining read counts per amplicon, and detecting exon copy numbers and whole gene deletions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing approaches are used, then small variants can be detected, but large rearrangements such as exon level copy number variations cannot be detected
Solution Approach 1:
The NGS assay is designed to perform multiple detection functions simultaneously - it can detect small variants (single nucleotide mutations and small insertions/deletions) as well as large rearrangements (exon level copy number variations, exon deletions, and exon duplications) within a single testing platform, eliminating the need for separate MLPA tests
2Device complexity
If traditional sequencing approaches are used, then the testing process is simple, but additional tests such as MLPA are required to detect large rearrangements
Solution Approach 1:
The invention merges the detection capabilities of traditional sequencing and MLPA into a single NGS assay. By combining the primer pool design with copy number analysis algorithms, the system integrates small variant detection and large rearrangement detection into one unified workflow, improving overall testing efficiency without requiring separate MLPA procedures
3Adaptability or versatility
If NGS assay is designed to detect both small mutations and large rearrangements, then comprehensive detection is achieved, but the assay complexity increases
Solution Approach 1:
The assay employs a segmented approach by dividing the BRCA1 and BRCA2 genes into multiple amplicons that cover different exons. Each amplicon can be independently analyzed for both sequence variants and copy number variations. This segmentation allows the complex detection task to be broken down into manageable units while maintaining comprehensive coverage of the entire gene region
Solution Approach 2:
The invention utilizes changes in read depth parameters across different amplicons to detect copy number variations. By monitoring the relative abundance of reads mapping to different amplicons and comparing against expected distributions, the system can infer exon deletions or duplications without requiring complex additional assays, thus managing assay complexity through parameter-based detection
4Reliability
If the NGS assay is to be compatible with FFPE samples, then clinical relevance is improved, but detection sensitivity for large rearrangements becomes more challenging
Solution Approach 1:
The assay incorporates preliminary quality control measures and normalization steps that are specifically optimized for FFPE samples. By pre-calibrating the system to account for FFPE-specific artifacts and degradation patterns, the assay maintains high detection sensitivity for large rearrangements even in clinically relevant FFPE samples, thus resolving the conflict between sample compatibility and detection precision
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the detection of both small mutations and large rearrangements, including exon deletions and duplications, in a single assay with high sensitivity, compatible with FFPE samples, improving diagnostic capabilities for breast and ovarian cancers.
Implementation Method 1
amplifying a nucleic acid sample in the presence of a primer pool to produce a plurality of amplicons
Implementation Method 2
sequencing the amplicons to generate a plurality of reads
Data Source
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AI summary
A method for detecting large rearrangements in BRCA1 and BRCA2 genes includes amplifying a nucleic acid sample in the presence of a primer pool to produce amplicons, where the primer pool includes target specific primers targeting regions of exons of the BRCA1 and BRCA2 genes. The method further includes sequencing the amplicons to generate a plurality of reads, mapping the reads to a reference sequence, determining a number of reads per amplicon for the amplicons associated with the exons of the BRCA and the BRCA2 genes, determining exon copy numbers for the exons of the BRCA1 and BRCA2 genes based on the number of reads per amplicon, detecting an exon deletion or duplication based on the exon copy numbers, and detecting a whole gene deletion of the BRCA1 or BRCA2 gene based on the number of reads per amplicon associated with the exons of the BRCA1 and BRCA2 genes.