Nanopore Multi-Pass Sequencing via Concatemer Consensus
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Solution Overview
Problem
Conventional single molecule sequencing methods, such as nanopore sequencing, suffer from high error rates, which limit the accuracy of sequence determination and require improved methods to enhance base-call accuracy and sequencing efficiency.
Innovation Solution
A multi-pass sequencing method using concatemer nucleic acid molecules with multiple copies of a target sequence separated by non-target sync sequences, allowing for nanopore sequencing of at least three copies to generate a multi-pass sequence dataset, which is then used to determine the target sequence with improved alignment and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If single molecule sequencing is used to achieve rapid sequencing with long read lengths, then sequencing speed and read length are improved, but base-call accuracy deteriorates to Q5-Q7 range
Solution Approach 1:
The patent combines multiple single-pass sequencing reads into a multi-pass consensus approach. By sequencing the same target region multiple times and merging the results through consensus calling, the method achieves high accuracy (Q30-Q40) while preserving the long read length advantage of single molecule sequencing.
Solution Approach 2:
The patent implements periodic re-sequencing of the same target region through multiple passes. The concatemer structure enables the polymerase to repeatedly translocate through the nanopore, generating multiple reads of the same sequence that can be combined to improve accuracy.
2Productivity
If multiple nanopores are used in parallel to increase sequencing throughput, then productivity is improved, but noise variability and measurement precision deteriorate
Solution Approach 1:
The patent creates multiple copies of the target sequence within a single concatemer molecule. This allows multiple reads to be generated from one molecule as it passes through a single nanopore multiple times, eliminating the need for multiple nanopores while maintaining high throughput and consistent signal quality.
3Device complexity
If conventional single-pass sequencing is used to maintain simplicity, then device complexity is reduced, but sequence accuracy deteriorates due to high error rates
Solution Approach 1:
The patent incorporates synchronization sequences at known positions within the concatemer structure before sequencing begins. These pre-placed marker sequences enable alignment and consensus calling during data analysis, improving accuracy without requiring complex real-time sequencing control mechanisms.
4Quantity of substance
If rolling circle replication is used to generate concatemers, then quantity of target copies is improved, but device complexity and manufacturing complexity increase
Solution Approach 1:
The patent utilizes the natural rolling circle replication capability of certain polymerases to automatically generate concatemer structures from circularized target DNA. This self-organizing process requires minimal external intervention and standard molecular biology reagents, making the method accessible without specialized equipment.
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 higher accuracy in sequence determination, reaching Q6, Q10, Q20, Q30, or better, while maintaining the advantages of long read lengths and speed, reducing noise variability from different nanopores and environmental factors.
Implementation Method 1
nanopore sequencing at least three copies of the target sequence in the concatemer, thereby obtaining a multi-pass sequence dataset
Data Source
AI summary
Improved single molecule sequencing methods, compositions, and devices, are provided. In a first aspect, the present invention provides a multi-pass method of sequencing a target sequence using nanopore sequencing, the method comprising: i) providing a non-naturally occurring concatemer nucleic acid molecule comprising a plurality of copies of the target sequence; ii) nanopore sequencing at least three copies of the target sequence in the concatemer, thereby obtaining a multi-pass sequence dataset, wherein the multi-pass sequence dataset comprises target sequence datasets for the at least three copies of the target sequence; and iii) using the multi-pass sequence dataset to determine the target sequence.