Correcting Deamination Errors in Cell-Free Nucleic Acid Sequencing
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Solution Overview
Problem
Current methods for detecting cancer through cell-free nucleic acids in bodily fluids face challenges due to low nucleic acid concentrations and the need for processing to achieve homogenous forms suitable for sequencing, which can introduce deamination-induced sequencing errors.
Innovation Solution
A method involving contacting nucleic acids with a protein having 5′-3′ polymerase and 3′-5′ exonuclease activity to generate double-stranded blunt-ended molecules, linking them to adapters, amplifying, and sequencing, while identifying variant nucleotides by analyzing sequence data to distinguish between real variations and deamination-induced errors based on context and proximity to ends.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cell-free nucleic acids are processed to achieve homogenous forms suitable for sequencing, then sequencing capability is improved, but deamination-induced sequencing errors are introduced
Solution Approach 1:
The patent applies preliminary action by performing end-repair processing on nucleic acid fragments before sequencing to generate blunt-ended molecules. This preprocessing step creates uniform blunt-ended nucleic acids that are then subjected to controlled deamination conditions, allowing subsequent identification and correction of deamination-induced errors through sequence analysis comparing expected versus observed nucleotide patterns
Solution Approach 2:
The patent converts the harmful deamination-induced sequencing errors into a detectable pattern by analyzing the specific context and proximity of C-to-T and G-to-A variations. By identifying that deamination errors occur predictably near fragment ends and in specific nucleotide contexts, the method transforms these errors into identifiable markers that can be distinguished from true biological variants, thereby converting a reliability problem into a solvable detection challenge
2Ease of operation
If nucleic acid concentration in bodily fluids is low, then non-invasive detection advantage is maintained, but detection sensitivity is reduced
Solution Approach 1:
The patent applies parameter changes by modifying the physical state and structure of low-concentration nucleic acid fragments through end-repair processing. By converting fragmented nucleic acids with overhangs into blunt-ended molecules, the method creates a uniform substrate that enhances subsequent sequencing efficiency and signal quality, thereby improving detection sensitivity without requiring higher nucleic acid concentrations or more invasive sampling
3Reliability
If deamination-induced errors are identified and corrected, then sequence accuracy is improved, but analysis complexity is increased
Solution Approach 1:
The patent implements feedback by using the observed sequence data to inform the identification and correction of deamination-induced errors. The method compares observed nucleotide variations against expected patterns based on fragment end proximity and nucleotide context, using this feedback loop to distinguish true biological variants from artifacts. This systematic feedback mechanism improves sequence accuracy while managing analysis complexity through rule-based discrimination
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 accuracy of identifying cancer-associated variant nucleotides by correcting for deamination-induced errors, improving the reliability of cancer detection from cell-free nucleic acids.
Implementation Method 1
contacting a population of nucleic acids comprising double-stranded molecules with single-stranded overhangs at one or both ends with a protein having 5'-3' polymerase activity and a 3'-5' exonuclease activity, wherein the protein digests 3' overhangs and fills in 5' overhangs
Implementation Method 2
contacting a population of nucleic acids comprising double-stranded molecules with single-stranded overhangs at one or both ends with a protein having 5'-3' polymerase activity and a 3'-5' exonuclease activity, wherein the protein digests 3' overhangs and fills in 5' overhangs with complementary nucleic acids
Data Source
AI summary
Sequencing nucleic acids can identify variations associated with presence, susceptibility or prognosis of disease. However, the value of such information can be compromised by errors introduced by or before the sequencing process including preparing nucleic acids for sequencing. Blunting single-stranded overhangs on nucleic acids in a sample can introduce deamination-induced sequencing errors. The disclosure provides methods of identifying and correcting for such deamination-induced sequencing errors and distinguishing them from real sequence variations.