MoCODE Barcode Multiplex PCR Library Construction
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Solution Overview
Problem
Current methods for constructing targeted enrichment libraries based on PCR are inefficient due to non-specific amplification issues, particularly in targeted methylation sequencing, where primer dimers and complex multimers are difficult to remove, leading to reduced sequencing quality and increased operational complexity.
Innovation Solution
The method involves using MoCODE barcodes, which are specific overhanging nucleotide sequences added to PCR products, allowing for efficient ligation with complementary sequencing adaptors, thereby reducing non-specific amplification and improving the specificity of the sequencing library construction process through sticky end ligation, thereby enhancing sequencing data quality and reducing operational time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional TA ligation or blunt end ligation is used in multiplex PCR library construction, then the ligation process is simple, but non-specific amplification products cannot be well removed and sequencing quality deteriorates
Solution Approach 1:
The patent divides the ligation process into two distinct stages: first, sticky-end ligation of adapters to PCR products; second, selective amplification using barcodes. This segmentation allows non-specific products to be identified and removed through barcode-based selection, while maintaining the simplicity of the overall workflow.
Solution Approach 2:
The patent introduces MoCODE barcodes as intermediary elements that carry specific identification information. These barcodes act as mediators between the PCR amplification step and the sequencing step, enabling selective enrichment of specific products while eliminating non-specific amplification artifacts.
2Manufacturing precision
If multiple PCR steps are performed for targeted enrichment, then enrichment specificity is improved, but operational complexity and time increase
Solution Approach 1:
The patent merges multiple functions into a single multiplex PCR reaction: targeted amplification, barcode tagging, and enrichment selection are all performed in one step. This consolidation maintains high enrichment specificity while dramatically reducing operational complexity compared to multiple sequential steps.
Solution Approach 2:
The patent designs a universal multiplex PCR system that can handle multiple target regions simultaneously through a single reaction. The universal primer sets and barcode system enable one reaction to perform what would traditionally require multiple specialized reactions, reducing both time and complexity.
3Measurement precision
If bisulfite treatment is applied to DNA for methylation sequencing, then methylation detection accuracy is improved, but primer dimer formation and non-specific amplification increase
Solution Approach 1:
The patent converts the harmful effect of bisulfite-induced sequence changes into a beneficial feature by designing primers that specifically target the converted sequences. The MoCODE barcodes are designed to work with the bisulfite-treated DNA, transforming the problematic sequence changes into reliable binding sites for specific amplification.
Solution Approach 2:
The patent introduces MoCODE barcodes as intermediary elements that mediate between the bisulfite-treated template and the amplification process. These barcodes provide specific binding sites that prevent primer dimer formation and non-specific amplification, while maintaining compatibility with the bisulfite-modified DNA sequences.
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 significantly reduces non-specific product formation, improves ligation efficiency, and simplifies the library construction process, resulting in higher-quality sequencing data with a 40-50% reduction in manual operation time and 30-40% reduction in overall construction time compared to existing methods.
Implementation Method 1
using the MoCODE barcodes to efficiently ligating the amplification product to sequencing adaptors comprising MoCODE barcode decoding sequences
Implementation Method 2
the MoCODE barcodes refer to overhanging single-stranded nucleotide sequences constituting two sticky ends of an obtained PCR product after the multiplex PCR product is digested with a specific endonuclease
Data Source
AI summary
A method for constructing a multiplex PCR library for high-throughput targeted sequencing: first acquiring a targeted DNA product by means of a high-specificity multiplex PCR reaction, and then digesting with a specific endonuclease, such that a specific molecular barcode is produced at the tail end of the PCR product; thus, the library construction process is more efficient, and the accuracy and sequencing depth of the obtained data are also ensured.