Surface-Anchored Second-Strand Synthesis for DNB Sequencing
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Solution Overview
Problem
The quality of second-strand sequencing in DNB paired-end sequencing is poor, characterized by high Lag%, low Q30%, and fast signal decrease, primarily due to the instability of Phi29 polymerase and the need for effective removal of residual polymerase and blocking agents, which affect the synthesis of the second strand.
Innovation Solution
A synthesis method for the second-strand in DNB paired-end sequencing that anchors a second-strand synthesis primer to the surface of a sequencing chip, using a modified primer with a bridge molecule, and employs Bst3.0 polymerase for stable synthesis, followed by digestion and removal of DNB using specific markers to ensure high-quality template production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Phi29 polymerase is used for second-strand synthesis, then the synthesis can be performed, but the enzyme activity decreases and stability is poor, resulting in high Lag% and low Q value
Solution Approach 1:
The patent changes the enzyme parameter from Phi29 polymerase to Bst3.0 polymerase, which has superior stability and activity. This parameter change resolves the contradiction by selecting an enzyme that maintains both high reliability (stability) and productivity (synthesis efficiency) throughout the second-strand synthesis process.
2Ease of manufacture
If strand displacement reaction is used to remove first-strand sequencing strand, then the template can be released, but residual Phi29 polymerase and blocking agents remain, affecting sequencing quality
Solution Approach 1:
The patent extracts and removes harmful factors (residual Phi29 polymerase and blocking agents) through a specific elution step using formamide. This extraction process eliminates the harmful substances that would otherwise remain in the template and affect subsequent sequencing quality, while still achieving efficient template release.
Solution Approach 2:
The patent introduces formamide as an intermediary elution reagent to selectively remove residual polymerase and blocking agents. This intermediary substance facilitates the removal of harmful factors without interfering with the DNB template structure, allowing clean template release and preparation for high-quality sequencing.
3Quantity of substance
If excessive strand displacement is performed, then more second-strand templates are generated, but residual Phi29 polymerase and blocking of ddNTPs increase, decreasing sequencing quality
Solution Approach 1:
The patent extracts and removes residual Phi29 polymerase and blocking agents through formamide elution after second-strand synthesis. This extraction prevents the harmful factors from accumulating and interfering with subsequent sequencing, allowing adequate template quantity while maintaining high sequencing quality.
Solution Approach 2:
The patent changes the synthesis parameters by using Bst3.0 polymerase instead of Phi29, which reduces excessive strand displacement and minimizes residual polymerase activity. This parameter change ensures adequate template generation without the harmful effects of over-synthesis.
4Reliability
If novel elution reagent is screened to increase elution time, then residual polymerase is effectively removed, but the sequencing efficiency is reduced
Solution Approach 1:
The patent optimizes the elution parameter by selecting formamide as the elution reagent with an optimized concentration and time range. This parameter optimization achieves effective residual polymerase removal while minimizing the elution time required, thus maintaining high sequencing efficiency without sacrificing reliability.
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
The method stabilizes the second-strand synthesis process, resulting in higher quality sequencing with improved Total Reads, Q30%, and ESR%, and reduced Lag%, enhancing the overall sequencing performance.
Implementation Method 1
an end of the second-strand synthesis primer has a modification, and is conjugated with a bridge molecule on the surface of the sequencing chip by means of the modification
Implementation Method 2
the described synthesis method uses a Bst3.0 polymerase for the second-strand synthesis
Implementation Method 3
using at least one of the following elution reagents to remove the first-strand sequencing strand: an organic denaturant, a 3'-5' exonuclease and NaOH; preferably, the organic denaturant is formamide
Implementation Method 4
after the second-strand synthesis, the DNB is digested and removed using the digestion marker
Data Source
Figure 1
Figure 2(A)~2(D)
AI summary
The present invention provides a synthesis method for a second strand in DNB paired-end sequencing, a sequencing method and a related product. The synthesis method for the second strand comprises: after a first-strand sequencing on the DNB is completed, performing a second-strand synthesis using a second-strand synthesis primer, wherein the second-strand synthesis primer is a primer capable of being anchored on the surface of a sequencing chip. Second-strand synthesis is performed by the second-strand synthesis primer anchored to a surface of a sequencing carrier, so that the process of the second-strand synthesis is relatively more stable, and the synthesized second-strand template strand is anchored to the sequencing carrier and subsequently the second-strand template strand anchored to the sequencing carrier is sequenced, so that a relatively higher quality of the second-strand sequencing can be obtained.