Sequencing Library Equidirectional Concatemer Error Correction
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Solution Overview
Problem
Current second-generation sequencing technologies face challenges in accurately determining DNA sequences due to high error rates, particularly when dealing with small amounts of degraded DNA from peripheral blood or ancient samples, limiting the detection of infrequent mutations and comprehensive genome analysis.
Innovation Solution
A sequencing library is created using an equidirectional alternating concatemer structure with a tag sequence linked to the DNA to be tested, allowing for accurate amplification and sequencing through rolling circle replication, which enables independent error correction and ultra-accurate mutation detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If second-generation sequencing technology is used to determine DNA sequences, then high-throughput sequencing can be achieved, but sequencing errors occur at about 1% rate which hinders accurate detection of infrequent mutations
Solution Approach 1:
The invention segments the DNA sequencing process into multiple independent replicates, where the same DNA template is sequenced multiple times. By dividing the sequencing task into separate replicates rather than relying on a single high-throughput run, the method enables error correction through comparison of results across replicates, thereby improving accuracy while maintaining throughput.
Solution Approach 2:
The invention implements a feedback mechanism by using breakpoint information from random DNA breaks to identify and correct sequencing errors. The system uses labels added to DNA templates before PCR amplification to track which DNA molecules derive from the same template, allowing errors to be detected and rectified by comparing sequences across multiple replicates.
2Measurement precision
If labels are added to DNA templates before PCR amplification to enable error correction, then determination accuracy improves, but the method is limited to small genomes or a small number of target genes due to required high sequencing depth
Solution Approach 1:
The invention segments the genome into multiple independent libraries, each prepared with labels and subjected to separate sequencing replicates. This segmentation allows the error correction method to be applied effectively to each segment without requiring prohibitively high sequencing depth across the entire genome, thereby enabling comprehensive genome-wide analysis.
Solution Approach 2:
The invention performs preliminary labeling of DNA templates before PCR amplification and sequencing. By adding labels to DNA templates in advance, the method enables subsequent error correction through breakpoint information without requiring re-labeling or additional steps during sequencing, making the approach feasible for large-scale genome analysis.
3Reliability
If non-amplification library building method is used to avoid PCR amplification errors, then amplification errors are eliminated, but the method cannot effectively handle small amounts of degraded DNA from peripheral blood or ancient samples
Solution Approach 1:
The invention extracts and removes the harmful PCR amplification step from the library preparation process by using a non-amplification method. Instead of relying on PCR to generate sufficient DNA from limited templates, the method directly prepares libraries from degraded DNA samples, eliminating amplification errors while maintaining effectiveness with small amounts of DNA through optimized library construction protocols.
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 effectively reduces sequencing errors and allows for precise determination of DNA sequences, even with limited DNA samples, enhancing the detection of infrequent mutations and enabling accurate analysis of whole genomes without relying on high sequencing depth.
Implementation Method 1
a tag sequence is linked to a DNA to be tested
Implementation Method 2
allowing for accurate amplification and sequencing through rolling circle replication
Data Source
AI summary
The present invention provides a sequencing library, and the sequencing library has an inserted fragment which is an equidirectional alternating concatemer of a sequence to be tested and a tag sequence. The present invention further provides a method for preparing the sequencing library. The present invention also provides a sequencing method. The sequencing library and sequencing method as provided in the present invention are capable of removing DNA amplification errors and sequencing errors under any sequencing depths, so that mutations of DNA molecules could be ultra-accurately determined. The sequencing library of the present invention is suitable for construction of a sequencing library of trace short DNA fragments and even of single-strand DNAs.