Tracer DNA Reference for Cell-Free DNA Quantification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for monitoring transplant rejection using donor-derived cell-free DNA (dd-cfDNA) face inaccuracies due to variable background cfDNA levels, leading to false negative or false positive results, especially in cases of viral infections or other factors affecting total cfDNA levels.
Innovation Solution
A method involving the use of Tracer DNA compositions added to biological samples for isolating and quantifying total and donor-derived cell-free DNA through targeted amplification and high-throughput sequencing, allowing for the determination of dd-cfDNA levels relative to a threshold value adjusted by total cfDNA levels, thereby improving the accuracy of transplant rejection detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If dd-cfDNA is reported as a percentage of total cfDNA, then the method is simple to implement, but the measurement precision deteriorates due to variable background cfDNA levels causing false negative or false positive results
Solution Approach 1:
The patent introduces an internal reference genome as an intermediary standard to mediate the quantification of dd-cfDNA. By comparing dd-cfDNA levels against a known reference genome in the same sequencing reaction, the method establishes a reliable baseline that corrects for variable background cfDNA levels, thereby improving measurement precision without significantly complicating the workflow
Solution Approach 2:
The patent changes the quantification parameter from a simple percentage ratio to an absolute concentration measurement normalized to an internal reference. This parameter transformation allows the method to account for variations in total cfDNA background levels while maintaining ease of implementation through standard sequencing workflows
2Productivity
If a fixed threshold value is used for dd-cfDNA to determine rejection, then the determination process is quick and simple, but the reliability deteriorates when total cfDNA levels are atypically high or low
Solution Approach 1:
The patent implements a dynamic thresholding approach where the rejection threshold is adjusted based on the measured total cfDNA level in each sample. When total cfDNA is atypically high or low, the threshold automatically adapts to maintain appropriate sensitivity and specificity, ensuring reliable rejection detection while preserving rapid determination through algorithmic adjustment rather than manual intervention
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
This approach enhances the precision of transplant rejection monitoring by accounting for variable background cfDNA levels, reducing false interpretations and providing a more reliable assessment of rejection risk.
Implementation Method 1
sequencing the amplification products by high-throughput sequencing to generate sequencing reads
Implementation Method 2
performing targeted amplification at 100 or more different target loci in a single reaction volume using 100 or more different primer pairs
Data Source
AI summary
The present disclosure provides methods for quantifying the amount of total cell-free DNA in a biological sample, comprising: isolating cell-free DNA from the biological sample, wherein a first Tracer DNA composition is added before or after isolation of the cell-free DNA; performing targeted amplification at 100 or more different target loci in a single reaction volume using 100 or more different primer pairs; sequencing the amplification products by high-throughput sequencing to generate sequencing reads; and quantifying the amount of total cell-free DNA using sequencing reads derived from the first Tracer DNA composition.