Cell-Free DNA Hydroxymethylation Profiling for Tissue Assignment
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Solution Overview
Problem
Current methods for analyzing cell-free DNA, particularly for epigenetic analysis, face challenges in detecting rare DNA modifications like 5-hydroxymethylcytosine (5hmC) with high efficiency and selectivity, as bisulfite sequencing cannot distinguish between 5mC and 5hmC, and existing methods lack precision in assigning tissue of origin.
Innovation Solution
A method utilizing hydroxymethylation profiles to probabilistically assign tissue of origin by comparing a sample's hydroxymethylation profile with a reference data set of tissue-specific gene loci, analyzing hydroxymethylation biomarkers to identify similar patterns and assign tissue type.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite sequencing is used to detect DNA methylation, then methylation profiles can be generated, but 5mC and 5hmC cannot be distinguished
Solution Approach 1:
The patent segments the detection process into two distinct pathways: one for detecting 5mC (using bisulfite sequencing) and another for detecting 5hmC (using oxidative enrichment followed by sequencing). This segmentation allows each method to optimize for its specific target modification, resolving the inability to distinguish between the two modifications when using a single bisulfite sequencing approach.
2Measurement precision
If cell-free DNA samples are analyzed for epigenetic modifications, then tissue of origin information can be obtained, but the small sample size and low DNA concentration make detection difficult
Solution Approach 1:
The patent applies preliminary enrichment of 5hmC-containing DNA fragments from cell-free DNA samples before sequencing. By performing oxidative treatment and affinity enrichment prior to sequencing, the method concentrates the rare 5hmC modifications from the limited cfDNA sample, enabling sufficient signal for accurate tissue assignment despite the small initial DNA quantity.
Solution Approach 2:
The patent introduces an intermediary enrichment step using oxidative treatment and affinity capture as a mediator between the cfDNA sample and final sequencing analysis. This intermediary process selectively isolates 5hmC-containing fragments, amplifying the signal from the limited starting material and enabling precise tissue of origin determination.
3Measurement precision
If 5hmC detection efficiency is increased to identify rare residues, then more 5hmC sites are detected, but selectivity may decrease leading to false positives
Solution Approach 1:
The patent employs parameter changes in the form of controlled oxidative treatment conditions (using TET enzymes or chemical oxidants under specific conditions) to selectively convert 5mC to 5hmC or directly detect 5hmC. By carefully controlling oxidation parameters, the method achieves high sensitivity for detecting rare 5hmC residues while maintaining selectivity through the specific biochemical properties of the oxidation reaction that distinguishes 5hmC from other DNA modifications.
Data Source
AI summary
A method is provided for probabilistically assigning a tissue of origin to a nucleic acid in a sample, e.g., DNA in a cell-free fluid sample obtained from a human subject. A hydroxymethylation profile is generated for the sample DNA and then compared across a reference data set of hydroxymethylation profile vectors, where each hydroxymethylation profile vector identifies the hydroxymethylation profile at a specific reference locus, the tissue-specific gene associated with the reference locus, and the tissue with which the gene and reference locus are associated. A tissue of origin can be probabilistically assigned to the sample nucleic acid using the results of the comparison. Other methods of use are also provided.