BRCA1/BRCA2 Copy Number Detection Using Sample ID Amplicons
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Solution Overview
Problem
Existing methods for detecting gene level copy number variations in BRCA1 and BRCA2 genes struggle to accurately distinguish between deletions and amplifications, often misclassifying imbalances as deletions.
Innovation Solution
Utilizing sample ID amplicons as a normal copy number anchor, the method employs targeted sequencing panels and next-generation sequencing to analyze BRCA1 and BRCA2 genes, incorporating DNA barcodes and adapters for accurate detection of whole gene copy number variations, including situations where both genes are affected.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing methods are used to detect copy number variations, then detection can be performed, but accuracy is insufficient leading to misclassification of deletions and amplifications
Solution Approach 1:
The patent introduces sample ID amplicons as intermediary reference markers that are distributed across different chromosomes. These amplicons serve as a mediator to calculate normalized read counts for BRCA1 and BRCA2, enabling accurate distinction between deletions and amplifications by providing a baseline for expected copy number ratios.
Solution Approach 2:
The patent changes the detection parameter from simple read count comparison to normalized read count ratio calculation. By dividing read counts for BRCA1/BRCA2 by the geometric mean of sample ID amplicon read counts, the method transforms raw sequencing data into normalized ratios that accurately reflect copy number variations.
2Ease of operation
If simple read count comparison is used, then the detection process is simple, but it cannot distinguish between deletions and amplifications
Solution Approach 1:
The patent segments the detection process into distinct functional components: (1) amplification of target regions including BRCA1/BRCA2 and sample ID regions, (2) sequencing to generate read counts, (3) calculation of normalized read counts using sample ID amplicons as references, and (4) determination of copy number variations. This segmentation maintains operational simplicity while improving precision.
Solution Approach 2:
The patent uses sample ID amplicons as reference copies that represent normal copy number. By comparing the read counts of BRCA1/BRCA2 to these reference copies, the method creates a normalized ratio that preserves the ability to distinguish between deletions (ratio < 1) and amplifications (ratio > 1).
3Reliability
If previous methods are used, then detection can be performed on tumor samples, but specificity is limited to 71.4% for somatic and 80% for germline samples
Solution Approach 1:
The patent incorporates feedback through the calculation of normalized read counts, where the sample ID amplicon read counts continuously inform the normalization process. This feedback mechanism adjusts the detection criteria dynamically, achieving 100% specificity for both somatic and germline samples by comparing actual ratios against expected thresholds.
Data Source
AI summary
Methods and systems for detecting gene level copy numbers for BRCA1 and BRCA2 genes include amplifying a nucleic acid sample in a presence of a primer pool to produce a plurality of amplicons. The primer pool may include target-specific primers targeting regions of exons of the BRCA1 and BRCA2 genes and sample ID regions. Overlapping amplicons cover the exons of the BRCA1 and BRCA2 genes. Sample ID amplicons are generated for targeted sample ID regions. The amplicons are sequenced to produce sequence reads. The sequence reads are mapped to a reference genome. Determining whole gene copy numbers for the BRCA1 and BRCA2 genes is based on the number of reads per amplicon for the amplicons associated with the exons of the BRCA1 and BRCA2 genes, respectively, and the number of reads per amplicon for the sample ID amplicons associated with the sample ID regions.