Two-Pass Sequencing with 3' End Blocking for Lower Noise
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Solution Overview
Problem
Existing single-molecule sequencing technologies suffer from high error rates and interference signals, particularly in platforms like Helicos, PacBio, and MinION, which can be improved by reducing InDel errors and sequencing noise.
Innovation Solution
A two-pass sequencing method is employed, where a sequencing template is ligated to a chip surface, followed by blocking the 3' end of the generated sequencing strand after the first run to prevent interference, and then performing a second sequencing run to enhance accuracy through mutual correction of the sequencing results.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If repeated sequencing is performed to reduce error rates, then sequencing accuracy is improved, but sequencing noise and interference signals increase
Solution Approach 1:
The sequencing process is divided into multiple independent passes, with each pass generating separate sequencing data. By segmenting the sequencing into distinct passes and selectively combining results, the method reduces noise while maintaining accuracy improvements from repeated sequencing.
Solution Approach 2:
The method applies different processing treatments to different portions of sequencing data. Specifically, it identifies and removes reads that span across the blocking region, while retaining and combining reads from non-blocking regions, thereby locally optimizing data quality in different segments of the sequencing output.
2Reliability
If Two-Pass sequencing is performed to reduce deletion errors, then error rate is reduced to less than 1%, but procedures become tedious and complicated
Solution Approach 1:
A blocking modification is introduced at the 3' end of the sequencing strand before the second sequencing pass. This preliminary action prevents polymerase from extending reads across the blocking region, thereby simplifying the data processing required in the second pass and reducing procedure complexity while maintaining low error rates.
Solution Approach 2:
The method modifies the chemical state of the sequencing strand by introducing a blocking group at the 3' end. This parameter change in the molecular structure prevents unwanted extension reactions, thereby simplifying the overall procedure while maintaining the reliability benefits of two-pass sequencing.
3Measurement precision
If multiple sequencing runs are performed to improve accuracy, then sequencing accuracy is improved, but time consumption increases
Solution Approach 1:
The blocking modification is applied once before the second sequencing pass, enabling parallel processing of multiple templates simultaneously. This preliminary action allows the system to prepare for accurate mutual correction without proportionally increasing time consumption, as the blocking prevents need for complex post-processing of each individual read.
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 significantly reduces sequencing noise and error rates by blocking the 3' end of the sequencing strand, allowing for improved accuracy and interference prevention in subsequent sequencing, thereby enhancing the overall sequencing results.
Implementation Method 1
the sequencing template being ligated to the surface of the chip through an adapter
Implementation Method 2
performing the first blocking on the 3' end of the first newly generated sequencing strand remaining on the surface of the chip to prevent the first newly generated sequencing strand from being extended
Data Source
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AI summary
An effective sequencing method, the method comprising: (1) performing first sequencing on a sequencing template on a chip surface, so as to facilitate obtaining first sequencing data by means of first newly generated sequencing strands being formed, the sequencing template being connected onto the chip surface by means of a sequencing adapter; (2) performing first blocking treatment on 3' ends of at least a portion of the first newly generated sequencing strands; and (3) performing second sequencing on the sequencing template, so as to facilitate obtaining second sequencing data by means of second newly generated sequencing strands being formed.