Library Construction Using 5' Phosphorylation for Dual Sequencing Platforms
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Solution Overview
Problem
Current library construction methods have low compatibility with both Illumina and MGI sequencing platforms, requiring separate workflows and increasing operational complexity and redundancy when transitioning between platforms.
Innovation Solution
A library construction method using primers and adaptors with 5' phosphorylation modifications, allowing for the creation of linear amplification libraries compatible with Illumina and cyclization libraries compatible with MGI, utilizing specific P5 and P7 truncated or full-length primers and adaptors to ensure compatibility and balance across sequencing platforms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If separate workflows are used for Illumina and MGI sequencing platforms, then platform-specific sequencing requirements are met, but operational complexity and workflow redundancy increase
Solution Approach 1:
The patent designs a universal library construction kit that works on both Illumina and MGI sequencing platforms. The kit includes platform-independent adapters and primers that can be used to construct libraries suitable for either platform, eliminating the need for separate workflows. This multi-functionality approach allows a single library construction protocol to serve dual purposes, reducing operational complexity while maintaining platform-specific sequencing requirements.
Solution Approach 2:
The patent employs 5' phosphorylation modification as a key parameter change to enable compatibility with both platforms. By adding a phosphate group to the 5' end of adapters and primers, the library construction process creates a universal interface that can be adapted to different sequencing platforms through subsequent cyclization or linear amplification steps. This chemical modification serves as a bridge between platform-specific requirements and a universal construction method.
2Adaptability or versatility
If PCR amplification is used to process Illumina libraries for MGI sequencing, then loading compatibility is achieved, but operational workflow and data redundancy increase
Solution Approach 1:
The patent performs the necessary library construction actions during the initial library preparation stage rather than as a subsequent processing step. By incorporating platform-independent adapters and 5' phosphorylation modifications during the original library construction, the patent eliminates the need for later PCR amplification processing. This preliminary action approach allows direct sequencing on either platform without requiring intermediate conversion steps, thereby reducing processing time while maintaining cross-platform compatibility.
3Productivity
If fluorescent signals are used for sequencing, then sequencing by synthesis is enabled, but signal overlap requires filters and base equalization scheduling
Solution Approach 1:
The patent applies local quality optimization through balanced index sequence design. By ensuring that index sequences have equal representation of different bases (base equalization) at specific local positions, the patent enables better separation of fluorescent signals during sequencing. This local optimization of sequence composition helps reduce signal overlap issues and simplifies the detection system requirements, allowing high-throughput sequencing with fewer filters and simpler scheduling.
Data Source
AI summary
The present application provides a library construction element, a kit and a library construction method compatible with double sequencing platforms. The library construction method comprises: performing library construction on target samples using primers or adaptors with a 5′ phosphorylation modification to obtain a linear amplification library with a 5′ phosphorylation modification, that is a linear library suitable for Illumina sequencing platform; or further cyclizing the linear amplification library with a 5′ phosphorylation modification to obtain a cyclization library suitable for the MGI sequencing platform.