Hairpin Adaptor PCR Sequencing for Low-Frequency Variant Detection
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Solution Overview
Problem
The existing next-generation sequencing (NGS) methods suffer from high error rates, particularly in detecting low-frequency variant genes, which is a challenge in precision medicine for accurately diagnosing and treating diseases like cancer.
Innovation Solution
A method involving the use of hairpin and Y-form adaptors to link to the ends of a double-stranded nucleic acid molecule, followed by PCR amplification cycles using specific primers, allows for the simultaneous amplification and sequencing of both forward and reverse strands of the target nucleotide sequence, enhancing accuracy and reducing sequencing costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional NGS methods are used, then sequencing can be performed, but the error rate is high (about 1%, minimum 0.1%) making it impossible to detect low-frequency variant genes
Solution Approach 1:
The invention segments the sequencing process into two independent directions: forward strand sequencing and reverse strand sequencing. By using hairpin adaptors that preserve both strands separately and sequencing each strand independently, the method achieves segmentation of the measurement process, allowing error correction through comparison of the two separate sequencing results.
Solution Approach 2:
The invention applies local quality by treating the forward and reverse strands differently during the sequencing process. Each strand is sequenced with its own optimized protocol and parameters, allowing local optimization of sequencing quality for each strand while maintaining the ability to cross-validate results for improved overall accuracy.
2Loss of information
If both forward and reverse strands are sequenced separately, then comprehensive sequence information can be obtained, but sequencing costs increase
Solution Approach 1:
The invention merges the sequencing of forward and reverse strands into a single integrated workflow. By using hairpin adaptors that keep both strands connected and using the same library preparation and sequencing run for both strands, the method combines what would traditionally be two separate sequencing experiments into one, reducing costs while obtaining complete sequence information from both strands.
Solution Approach 2:
The hairpin adaptor structure serves multiple functions simultaneously: it preserves both forward and reverse strands, provides sequencing priming sites for both directions, and enables both strands to be processed through the same library preparation protocol. This multi-functionality eliminates the need for separate processing workflows, reducing overall sequencing costs.
3Ease of manufacture
If conventional adaptor ligation is used, then nucleic acid molecules can be prepared for sequencing, but the structure does not preserve both forward and reverse strands for simultaneous analysis
Solution Approach 1:
The hairpin adaptor structure implements a nested doll configuration where one strand is folded back and nested within itself to form a hairpin loop. This nested structure preserves both the forward and reverse strands in a compact form, allowing both to be present in the same molecule and accessible for sequencing while maintaining ease of manufacture through standard ligation protocols.
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 improves the accuracy of nucleotide sequence determination by aligning complementary DNA strands and reduces sequencing costs by holding them together for analysis, thereby improving the precision of genetic sequencing.
Implementation Method 1
two complementary strands of DNA (i.e., forward strand and reverse strand) of the target nucleotide sequence are held together by the hairpin second adaptor
Implementation Method 2
a first primer, a second primer, and a DNA polymerase are brought in contact with the nucleic acid template under the conditions sufficient for a PCR amplification of the target nucleotide sequence
Data Source
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AI summary
Methods of amplifying and determining a target nucleotide sequence are provided. The method of amplifying the target nucleotide sequence includes the following steps. A first adaptor and a second adaptor are linked to two ends of a double-stranded nucleic acid molecule with a target nucleotide sequence respectively to form a nucleic acid template, in which the first adaptor includes a Y-form adaptor or a hairpin adaptor and the second adaptor is a hairpin adaptor. Then, a PCR amplification cycle is performed on the nucleic acid template to obtain a PCR amplicon of the target nucleotide sequence.