Highly Accurate DNA Sequencing with Complementary-Strand Consensus
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Solution Overview
Problem
Existing DNA sequencing methods struggle to accurately identify low-frequency mutations, such as those present in a few cell populations or individually generated mutations, due to sequencing errors and the need for specialized library preparation or sequencers, limiting their applicability to cancer tissues and environmental mutagen analysis.
Innovation Solution
A method that sequences DNA fragments, amplifies them through PCR to generate multiple reads, collects sequences from the same DNA region, and builds consensus between complementary strands without the need for tag sequences, simplifying library preparation and enhancing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If tag sequences are added to DNA fragments for accurate sequencing, then sequencing accuracy is improved, but library preparation complexity increases
Solution Approach 1:
The invention extracts and utilizes the inherent partial sequences of DNA fragments themselves as identification markers, removing the need for externally added tag sequences. This allows accurate tracking of DNA fragments through sequencing while simplifying library preparation procedures.
Solution Approach 2:
The DNA fragments serve their own identification function through their intrinsic partial sequences rather than requiring external tagging. The partial sequences naturally present in the fragments are used to identify and group reads originating from the same fragment, making the system self-sufficient.
2Reliability
If oxidative modification errors occur during library preparation, then sequencing accuracy deteriorates, but error identification becomes difficult without complementary strand information
Solution Approach 1:
The invention uses the complementary strand as an intermediary reference to identify and correct errors. By comparing reads from both complementary strands, oxidative modification errors that occur on only one strand can be distinguished from true mutations present in both strands.
Solution Approach 2:
The sequencing results from complementary strands provide feedback for error detection and correction. The consensus information from both strands allows identification of erroneous reads, enabling correction of sequencing errors and improvement of overall accuracy.
3Measurement precision
If single cell isolation is performed for mutation analysis, then low-frequency mutations can be identified, but time consumption and operational complexity increase
Solution Approach 1:
The invention segments the analysis by focusing on specific partial sequences of DNA fragments rather than requiring complete genome sequencing of single cells. This allows efficient identification of low-frequency mutations through targeted analysis of fragment subsets.
Solution Approach 2:
The method creates multiple copies (reads) of DNA fragments through PCR amplification and sequencing, then uses consensus building from these copies to identify true mutations. This approach to low-frequency mutation detection avoids the time-consuming single cell isolation process.
4Measurement precision
If read sequences from different DNA fragments are misrecognized as same fragment, then sequencing accuracy deteriorates, but tag sequences prevent this misrecognition
Solution Approach 1:
DNA fragments use their own inherent partial sequences for identification rather than requiring external tag sequences. The partial sequences naturally present in each fragment serve as unique identifiers, enabling accurate differentiation between fragments without adding system complexity.
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 achieves high sequencing accuracy for low-frequency mutations by collecting and aligning sequences from complementary DNA strands, reducing errors and enabling broader applicability across different sequencers and sample types.
Implementation Method 1
subjecting the fragments of the sample DNA to PCR to produce two or more amplified fragments for each of the fragments of the sample DNA
Data Source
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Figure 3A~3B
AI summary
Provided is a highly accurate sequencing method, specifically, a method for sequencing DNA, the method comprising: (1) preparing fragments of sample DNA; (2) subjecting the fragments of the sample DNA to PCR to produce two or more amplified fragments for each of the fragments of the sample DNA, and obtaining PCR products containing a plurality of amplified fragments; (3) sequencing the PCR products to generate one or more read sequences from the plurality of amplified fragments, and acquiring a plurality of read sequences for the plurality of amplified fragments; (4) collecting, from the plurality of read sequences acquired, read sequences containing sequence information on the same region in the sample DNA into a group to generate one or more groups of read sequences; and (5) building consensus of sequence information among read sequences included in each of the groups of read sequences.