Y-shaped Linker Design for Sequencing Index Hopping
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Solution Overview
Problem
Whole genome sequencing assays face high sequence index hopping and redundancy issues due to residual unengaged index linkers and rapid amplification of PCR-free libraries, leading to inaccurate data and increased sequencing costs.
Innovation Solution
A method using a novel linker combination of a reverse complementary Y-shaped linker and a high GC clamp linker is introduced, where the P7 end of the linkers is changed from 3' to 5' orientation and a high GC sequence is added to the 5' end to prevent sequence index hopping and redundancy, specifically designed for patterned flow cell technology platforms like NovaSeq.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR-free library preparation is used, then sequencing accuracy and sensitivity for rare mutation detection are improved, but sequence index hopping ratio increases
Solution Approach 1:
The patent divides the index linker into two separate indexes (first index and second index) positioned at different ends of the DNA fragment. This segmentation allows independent verification of each index, preventing index hopping from compromising overall sample identification accuracy.
Solution Approach 2:
The patent introduces a unique dual index system as an intermediary mechanism between the DNA sample and the sequencing platform. The combination of first and second indexes acts as a mediator that ensures accurate sample identification by requiring both indexes to match the expected pattern, thereby blocking the harmful effect of index hopping.
2Measurement precision
If dual indexes are used to solve index hopping, then detection accuracy is improved, but linker preparation and maintenance difficulty increases
Solution Approach 1:
The patent merges the first index and second index into a single dual-indexed linker structure that is ligated as one unit to the DNA fragment. This combining approach simplifies the preparation process by reducing the number of separate indexing steps while maintaining the accuracy benefits of dual indexing.
Solution Approach 2:
The dual-indexed linker serves multiple functions simultaneously: it provides sample identification through two indexes, maintains structural integrity during sequencing, and enables accurate data demultiplexing. This multi-functionality reduces the need for separate preparation steps for each function.
3Productivity
If patterned flow cell technology is used, then sequencing throughput is improved, but redundancy increases
Solution Approach 1:
The patent implements a feedback mechanism through dual index verification where the sequencing system checks both the first and second indexes against the expected pattern. This feedback loop identifies and filters out redundant or erroneous sequences, ensuring that only valid data is retained for analysis.
Solution Approach 2:
The patent changes the parameter of index structure from single-index to dual-index configuration. This parameter change enables the system to distinguish between true sequencing signals and redundant artifacts, thereby reducing effective redundancy while maintaining high throughput.
Data Source
AI summary
The present disclosure relates to a method for constructing a whole genome high-throughput sequencing library comprising the following steps: (1) extracting a sample gDNA; (2) fragmenting said sample gDNA by enzyme cleavage, filling ends of the gDNA and adding A base to the gDNA fragments to obtain an A-added gDNA; (3) connecting the A-added gDNA with a linker combination to obtain a connected produce, said linker combination comprises two parts: a Y-shaped reverse linker and a high GC clamp linker; (4) purifying said connected product to obtain a purified product; and (5) screening the fragment of said purified product to obtain a sequencing library. The present disclosure also relates to a kit for constructing a whole genome high-throughput sequencing library.