Homogeneous Multiple Tagging of Long DNA for Haplotype Phasing
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Solution Overview
Problem
Current methods for determining parental contributions to genomes, particularly in higher organisms, are inefficient and costly, and struggle with maintaining long DNA fragments for haplotyping, especially in clinical and metagenomics applications, lacking the ability to accurately assemble parental chromosomes and resolve variations in complex nucleic acids.
Innovation Solution
A method called Multiple Tagging (MT) introduces unique identifiers into long DNA fragments in a homogeneous reaction, allowing for the assembly of parental chromosomes without physical compartmentalization, using transposons or hairpin sequences to tag and amplify subfragments for accurate sequencing and haplotype phasing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If physical compartmentalization (nanodrops) is used to tag long DNA fragments, then tagging accuracy is improved, but device complexity and processing difficulty increase
Solution Approach 1:
The patent extracts the tagging process from the complex physical compartmentalization environment (nanodrops) and performs it in a simple homogeneous solution, eliminating the need for droplet generation and manipulation while maintaining tagging accuracy through molecular proximity effects
Solution Approach 2:
The patent replaces the mechanical system of physical compartmentalization (nanodrop generation, handling, and processing) with a chemical/biochemical system where tagging occurs in solution through molecular interactions, eliminating complex mechanical processing steps
2Measurement precision
If long DNA fragments (>100 kb) are processed for haplotyping, then haplotype phasing accuracy is improved, but processing feasibility and yield decrease
Solution Approach 1:
The patent segments long DNA fragments into smaller subfragments after tagging, allowing the tagged fragments to be processed through standard sequencing workflows while the tag sequences enable computational reconstruction of the original long fragment haplotypes
Solution Approach 2:
The patent introduces tag sequences as intermediary markers that are inserted into long DNA fragments, allowing these fragments to be tracked and assembled through standard short-read sequencing processes, thereby bridging the gap between long fragment information and short read processing capabilities
3Measurement precision
If cloning into BAC vectors is used for long fragment sequencing, then long-range haplotyping is achieved, but cost and processing time increase
Solution Approach 1:
The patent performs preliminary tagging of long DNA fragments with unique identifiers before sequencing, allowing direct assembly of haplotypes from sequencing data without requiring time-consuming BAC library construction, transformation, and colony screening steps
Solution Approach 2:
The patent uses tag sequences that can be copied and detected through standard sequencing methods, replacing the need for physical BAC clone isolation and characterization, thereby accelerating the haplotyping process while maintaining accuracy
4Adaptability or versatility
If metagenomic mixtures are analyzed, then microbial diversity information is obtained, but sequence assembly accuracy decreases due to complexity
Solution Approach 1:
The patent segments metagenomic DNA into tagged fragments, allowing each fragment to be tracked through the sequencing process and accurately assembled by matching tag sequences, thereby resolving the complexity of mixed microbial communities while maintaining assembly accuracy
Solution Approach 2:
The patent uses tag sequences as feedback markers that provide continuous information about fragment identity and origin throughout the sequencing and assembly process, enabling accurate reconstruction of parental chromosomes even in complex metagenomic mixtures
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
MT enables accurate haplotype phasing and variant calling at reduced costs, facilitating high-throughput sequencing and diagnostic applications by minimizing errors and requiring minimal DNA input, suitable for diploid and polyploid genomes, cancer diagnostics, and metagenomics.
Implementation Method 1
using transposons or hairpin sequences to tag and amplify subfragments for accurate sequencing and haplotype phasing
Implementation Method 2
tag and amplify subfragments for accurate sequencing
Data Source
Figure 1A
Figure 1B
Figure 2A
AI summary
The present invention provides methods and compositions for tagging long fragments of a target nucleic acid without using compartmentation, e.g. nanodrops. The method uses transposase and beads labelled with at least 1000 copies of the same tag. The tag is incorporated into the long fragments under conditions which promote the interaction of one type of tag and one long DNA fragment. The tagged fragments may be used for sequencing.