Bi-directional Nucleic Acid Sequencing via Template Nicking
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Solution Overview
Problem
Current nucleic acid sequencing methods lack efficient methods for bi-directional sequencing, which is essential for obtaining comprehensive sequence information from nucleic acid molecules, especially for longer templates or to validate sequence information independently.
Innovation Solution
The development of methods and systems that enable bi-directional sequencing by hybridizing primers to nucleic acid molecules linked to supports, extending these primers, introducing nicks, and degrading portions to generate single-stranded regions for sequencing in both forward and reverse directions, using techniques like ion-based sequencing and template-dependent nucleotide incorporation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If conventional single-direction sequencing methods are used, then the sequencing process is simple and quick, but comprehensive sequence information cannot be obtained from longer nucleic acid templates
Solution Approach 1:
The sequencing process is divided into two distinct directional reads (forward and reverse) from opposite ends of the template strand. This segmentation allows each read to cover a specific portion of the template, enabling complete sequence information to be obtained from longer templates by combining results from both directions.
Solution Approach 2:
The invention transitions from single-direction sequencing to bi-directional sequencing by introducing a new dimensional approach - sequencing from both the 5' and 3' ends simultaneously. This dimensional change enables comprehensive coverage of the entire template strand regardless of its length.
2Reliability
If bi-directional sequencing is implemented, then comprehensive sequence information and validation are achieved, but the sequencing process becomes more complex
Solution Approach 1:
The bi-directional sequencing approach provides inherent feedback mechanisms where the forward and reverse reads can be compared and validated against each other. This feedback loop enables independent validation of sequence information, improving reliability by allowing detection and correction of sequencing errors through cross-verification.
3Loss of information
If longer template strands are sequenced, then more comprehensive genetic information is obtained, but read length limitations prevent complete sequencing
Solution Approach 1:
The template strand is effectively segmented into two sequencing regions by initiating reads from both ends. The forward read starts from the 5' end and the reverse read starts from the 3' end, allowing each read to cover approximately half of the template length. This segmentation enables complete coverage of long templates even when individual read lengths are limited.
Solution Approach 2:
By adding the temporal/directional dimension of sequencing from opposite ends, the invention overcomes the physical limitation of fixed read length. The combination of forward and reverse reads creates complete coverage of the entire template strand, effectively extending the usable sequencing length beyond the limitations of individual read lengths.
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 enhances sequencing accuracy and throughput by allowing comprehensive sequence determination in both orientations, improving data consistency and reliability, particularly for longer sequences.
Implementation Method 1
nucleic acid synthesis is performed via serial incorporation of nucleotides in a template-dependent fashion, typically using a polymerase
Implementation Method 2
label-free methods of sequencing-by-synthesis have been developed, including so-called 'ion based' sequencing, wherein one or more byproducts of nucleotide incorporation can be detected electronically using chemically sensitive transistors
Data Source
AI summary
In some embodiments, methods for obtaining sequence information from a nucleic acid template linked to a support include hybridizing a first primer to a template strand linked to a support, sequencing a portion of the nucleic acid template, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid template, In some embodiments, the method further includes introducing a nick into a portion of the template strand that is hybridized to the extended first primer product, degrading a portion of the template strand from the nick using a degrading agent, where a portion of the extended first primer remains hybridized to an undegraded portion of the template strand, and sequencing at least some of the single-stranded portion of the extended first primer by synthesis.