Non-unique Barcodes for ctDNA Genotyping Assay Sensitivity
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Solution Overview
Problem
Current methods for analyzing circulating tumor DNA (ctDNA) in blood samples face challenges due to low DNA concentrations and high error rates in genotyping technologies, making it difficult to detect rare mutations with sufficient sensitivity and specificity.
Innovation Solution
The use of non-unique barcodes in combination with endogenous barcodes for ctDNA assays, which allows for high-precision and accurate analysis of multiple genomic regions from a single sample, thereby enhancing sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional genotyping technology is used to analyze ctDNA, then the analysis can be performed, but the sensitivity and specificity are insufficient due to low DNA concentration and high error rates
Solution Approach 1:
The invention segments the barcode identification process into two independent components: exogenous barcodes (added during library preparation) and endogenous barcodes (inherent in the DNA fragment ends). This segmentation allows the system to overcome the limitations of using either barcode type alone, particularly in low-input samples where one barcode type may be insufficient. By combining both barcode types, the system achieves higher measurement precision and reliability in detecting rare mutations in ctDNA.
2Measurement precision
If a large number of unique barcodes are used to track DNA fragments, then tracking accuracy improves, but the complexity of the assay increases
Solution Approach 1:
The invention merges two barcode systems (exogenous and endogenous) into a unified tracking approach. Instead of relying on a single complex barcode system with many unique identifiers, the system combines two simpler barcode types that together provide sufficient tracking accuracy. This merging reduces assay complexity while maintaining or improving measurement precision, as each barcode type can be independently optimized and validated.
Solution Approach 2:
The invention adds a second dimension to barcode identification by incorporating both exogenous barcodes (first dimension) and endogenous barcodes (second dimension). This dimensional approach allows the system to track DNA fragments with higher accuracy without requiring an exponentially larger number of unique barcodes in a single dimension. The combination of two dimensions with fewer options each is mathematically equivalent to or better than one dimension with many options, thereby reducing assay complexity.
3Measurement precision
If high coverage sequencing is performed on low-abundance ctDNA samples, then detection sensitivity improves, but the impact of sequencing errors increases
Solution Approach 1:
The invention introduces dual barcodes as intermediary identifiers that mediate between the DNA fragments and the sequencing process. These barcodes (both exogenous and endogenous) serve as intermediaries that allow the system to track and reconcile sequences from the same original DNA molecule across multiple sequencing reads. This intermediary system enables high coverage sequencing to be performed on low-abundance samples while providing a mechanism to distinguish true mutations from sequencing errors through barcode-based consensus building.
Solution Approach 2:
The invention implements a feedback mechanism where sequences are reconciled based on matching barcode combinations. The system uses the dual barcode information to feed back into the analysis process, allowing sequences with matching barcodes to be compared and reconciled. This feedback loop enables the system to identify and correct sequencing errors by comparing multiple reads of the same DNA molecule, thereby improving mutation detection sensitivity while mitigating the impact of sequencing errors.
Data Source
AI summary
The present disclosure involves ctDNA assays that interrogate many regions from a single sample with high precision and accuracy, while evaluating multiple forms of cancer-related genomic alterations including sequence mutations and structural alterations. The disclosure provides simplified yet robust methods that achieve high sensitivity and specificity by analyzing cancer genes using a limited pool of non-unique barcodes in combination with endogenous barcodes. Samples are captured and sequenced using high coverage next-generation sequencing to allow tumor-specific somatic mutations, amplifications, and translocations to be identified.