Barcoded Transposase Complex for High-Throughput Sequencing
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Solution Overview
Problem
Current second-generation sequencing technologies face limitations in obtaining accurate genomic information due to short read lengths and small span, leading to inefficient haplotype information retrieval, and the stLFR technology is restricted to single-sample library construction, resulting in wasted resources and costs for small genomic samples.
Innovation Solution
A barcoded transposase complex is developed, featuring a transposase recognition element with a specific structure and spacer region, allowing for mixed library construction of multiple samples using a single reaction tube, which improves library construction throughput and reduces costs by enabling high-throughput automation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional second-generation sequencing is used, then sequencing cost is reduced and throughput is increased, but read length is limited and haplotype information is lost
Solution Approach 1:
The patent embeds multiple levels of barcodes within the sequencing library structure. Molecular barcodes are incorporated during library construction, and sample barcodes are added during sequencing, creating a nested information system that preserves both long-range haplotype information and high-throughput sequencing capability
Solution Approach 2:
The patent adds the dimension of barcode sequences to the traditional sequencing approach. By incorporating molecular barcodes and sample barcodes as additional information dimensions, the system recovers haplotype information without compromising sequencing throughput
2Measurement precision
If stLFR technology is used for single-sample library construction, then long-fragment information is obtained, but sequencing resources are wasted and costs increase for small genomic samples
Solution Approach 1:
The patent combines multiple samples into a single library construction reaction by incorporating sample-specific barcodes. This merging approach allows parallel processing of multiple samples while maintaining the ability to distinguish and analyze long-fragment information for each sample separately
Solution Approach 2:
The barcoded transposase complex serves multiple functions: it fragments DNA, adds molecular barcodes for long-fragment tracking, and incorporates sample barcodes for multiplexing. This multi-functionality enables both high precision and high throughput in a single system
3Productivity
If mixed library construction for multiple samples is implemented, then sequencing resource utilization is improved and costs are reduced, but library construction complexity increases
Solution Approach 1:
The barcoded transposase complex performs self-identification and self-sorting through its embedded barcodes. During library construction, the complex automatically associates the correct molecular and sample barcodes with each DNA fragment, eliminating the need for complex external tracking and management systems
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 solution enables efficient mixed library construction for multiple samples, reducing complexity and costs, improving sequencing throughput, and allowing for accurate haplotype sequencing with reduced sample input, making it suitable for rare and low-biomass samples.
Implementation Method 1
a transposase is used for fragmenting the high-molecular-weight DNA
Data Source
AI summary
A barcoded transposase complex and an application thereof in high-throughput sequencing. Provided is a transposase recognition element, having the following structure: X(m)Y(f)N(n), in which X(m) represents a transposase recognition region of a double-stranded nucleic acid structure, Y(f) represents a spacer region of a single-stranded DNA structure, and N(n) represents a sample barcode of a single-stranded DNA structure. The high-molecular-weight DNA is processed using the barcoded transposase complex, to obtain a lot of barcoded DNA fragments. The barcoded DNA fragments obtained from each high-molecular-weight DNA are mixed to obtain a mixing sample. A carrier having a molecular barcode is adopted to capture. An exonuclease is adopted for processing, and then transposase is released. StLFR technology is adopted to construct a DNA library. The barcoded transposase complex can be applied to hybrid sequencing of a high-throughput sequencing platform.