Plasma ctDNA Enrichment Sequencing for Low-Frequency Mutation Detection
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Solution Overview
Problem
Current methods for detecting low-frequency mutations in circulating tumor DNA (ctDNA) in plasma are limited in sensitivity and specificity, particularly for early-stage cancer diagnosis, as they struggle to accurately identify mutations present at frequencies as low as 0.01%, requiring high-throughput sequencing and large sample volumes.
Innovation Solution
A method involving universal library TT-COLD PCR, probe-based enrichment, and hybridization capture followed by sequencing, combined with forward and reverse double-strand error correction, to enrich and sequence low-frequency mutations in plasma, utilizing a three-step enzymatic library construction and specific primer sequences for efficient mutation detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods (preMiDTM, CAPP-Seq) are used, then detection can be performed with standard sample volumes, but detection sensitivity is insufficient to accurately identify mutations at 0.01% frequency
Solution Approach 1:
The detection process is divided into multiple enrichment stages: first enrichment by COLD-PCR to increase mutation frequency from 0.01% to approximately 1%, followed by second enrichment by target area capture to further concentrate mutations. This segmented approach achieves high detection sensitivity while working with limited sample volumes (5-10 mL plasma).
Solution Approach 2:
The method performs preliminary enrichment of mutation-containing DNA fragments before sequencing through COLD-PCR and target area capture. This preliminary action concentrates the rare mutations to detectable levels, enabling accurate identification of 0.01% frequency mutations without requiring excessively large sample volumes.
2Measurement precision
If high-throughput sequencing is performed directly on plasma ctDNA, then comprehensive mutation information can be obtained, but the low abundance of ctDNA (0.01% frequency) results in insufficient detection sensitivity
Solution Approach 1:
The methodology segments the detection process into distinct enrichment phases (COLD-PCR enrichment, target area capture) followed by high-throughput sequencing. This segmentation ensures that mutations are sufficiently concentrated before sequencing, making the detection reliable even for early-stage cancers with very low ctDNA abundance.
Solution Approach 2:
The patent introduces intermediary enrichment steps (COLD-PCR and target area capture) between sample preparation and high-throughput sequencing. These intermediary processes act as mediators that amplify and concentrate the rare mutations, bridging the gap between low-abundance input material and the detection threshold required for reliable sequencing.
3Measurement precision
If Duplex Sequencing with UID label error correction is used, then sequencing errors can be corrected with detected mutation frequency of 10^-7, but the technique requires sequencing throughput higher than conventional sequencing
Solution Approach 1:
The patent applies partial error correction by focusing on correcting errors in enriched mutation regions rather than implementing full Duplex Sequencing across the entire genome. The enrichment steps concentrate mutations to high frequency, allowing conventional sequencing with basic error correction to achieve sufficient accuracy without the excessive throughput requirements of complete Duplex Sequencing.
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 method enables the accurate detection of low-frequency mutations with high sensitivity and specificity, reducing the required sample volume to 5-10 mL, and allows for simultaneous analysis of multiple samples, enhancing early screening and monitoring capabilities for various cancers.
Implementation Method 1
amplification and enrichment by universal library TT-COLD PCR
Implementation Method 2
enrichment and capture with probes, and amplification and sequencing of hybridization captured product
Data Source
AI summary
The present invention provides a low-frequency mutation enrichment sequencing method for free target DNA in plasma, comprising plasma DNA extraction and library construction, general library TT COLD PCR amplification enrichment, probe enrichment capture, PCR and sequencing of captured products, and positive and negatice double-strand error-correction low-frequency information analysis.