Double-Strand Sequencing via Invasion Primers
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Solution Overview
Problem
Traditional next-generation sequencing methodologies face challenges in detecting rare sequence variants due to polymerase errors and are laborious and expensive, requiring specialized equipment and expertise.
Innovation Solution
A method involving hybridizing invasion and sequencing primers to double-stranded polynucleotides attached to a solid support, generating invasion and sequencing strands, and detecting incorporated nucleotides to sequence both strands efficiently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional next generation sequencing methodologies are used, then millions of fragments can be sequenced simultaneously, but detection of rare sequence variants is compromised due to polymerase errors
Solution Approach 1:
The method segments the sequencing process by separately sequencing the forward and reverse strands of the polynucleotide. By generating independent sequencing reads from each strand and comparing them, the system can distinguish true rare variants from polymerase errors, thereby improving measurement precision while maintaining high productivity through parallel processing of multiple fragments
Solution Approach 2:
The invention applies local quality control by focusing on the comparison between forward and reverse strand sequences at each position. By examining local discrepancies between the two strands, the method can identify rare variants with high confidence while filtering out random polymerase errors, thus improving variant detection accuracy without sacrificing throughput
2Measurement precision
If Sanger sequencing methodology is used, then sequence validation is achieved, but the process becomes expensive and laborious requiring specialized equipment and expertise
Solution Approach 1:
The method creates a complementary copy of the sequence by sequencing both the forward and reverse strands. This copying approach provides validation similar to Sanger sequencing, as the two strands serve as mutual verification, but can be implemented using standard NGS equipment rather than specialized Sanger sequencing apparatus, thereby reducing device complexity and expertise requirements
3Productivity
If traditional NGS methods are used, then high throughput sequencing is achieved, but the process remains laborious and expensive
Solution Approach 1:
The invention merges the sequencing of forward and reverse strands into a single integrated workflow. By combining both sequencing reactions and analyzing them together, the method achieves comprehensive sequence validation in one process rather than requiring separate Sanger sequencing steps, thereby reducing labor and processing time while maintaining high throughput through parallel processing of multiple fragments
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 enhances sequencing accuracy and efficiency, improving the detection of rare variants while reducing costs and equipment requirements.
Implementation Method 1
extending the first invasion primer hybridized to the second strand with a polymerase, thereby generating a first invasion strand
Implementation Method 2
extending the second invasion primer hybridized to the first strand with a polymerase, thereby generating a second invasion strand
Implementation Method 3
detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in the first extension strand, thereby sequencing the first strand
Data Source
AI summary
Disclosed herein, inter alia, are methods for sequencing both strands of a double stranded nucleic acid fragment. Compositions and kits for use in the methods are also provided.