Cross-linked DNA Proximity Ligation for Genome Coverage
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Solution Overview
Problem
Next-generation sequencing technologies face challenges in achieving full genome coverage and preserving spatial-proximal contiguity information, leading to incomplete or inaccurate genomic data.
Innovation Solution
The method involves contacting cross-linked DNA molecules with restriction endonucleases to generate spatial-proximal digested ends, followed by ligation to create proximity-ligated DNA molecules, which are then fragmented to produce templates for short-range sequencing, preserving spatial-proximal contiguity information and achieving full genome coverage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If native genomic DNA is fragmented to produce nucleic acid templates for sequencing, then sequencing can be performed, but spatial-proximal contiguity information is lost
Solution Approach 1:
The patent applies preliminary action by cross-linking DNA molecules before fragmentation to preserve spatial-proximal contiguity information. The cross-linking step is performed in advance on intact chromatin, creating covalent bonds between spatially proximal DNA segments before any fragmentation occurs, thus preserving the spatial relationship information through subsequent processing steps
Solution Approach 2:
The patent uses proximity ligation as an intermediary mechanism to bridge spatially proximal DNA ends. By using ligase to join cross-linked DNA ends that are spatially proximal, the method creates chimeric molecules that serve as intermediaries carrying spatial relationship information, which can then be detected through sequencing
2Quantity of substance
If whole genome sequencing is performed to achieve full genome coverage, then comprehensive genomic data is obtained, but spatial-proximal contiguity preservation is compromised
Solution Approach 1:
The patent applies segmentation by dividing the genome into restriction fragments and performing proximity ligation on these segments. By digesting chromatin with restriction enzymes to create manageable fragments and then performing ligation on these segmented pieces, the method achieves both comprehensive coverage through multiple fragments and spatial information preservation through the ligation of spatially proximal segments
3Loss of information
If cross-linked DNA molecules are digested with restriction endonucleases and ligated to create proximity-ligated molecules, then spatial-proximal contiguity is preserved, but the process complexity increases
Solution Approach 1:
The patent merges multiple operations into an integrated workflow: cross-linking, restriction digestion, and proximity ligation are combined in a sequential manner within the same reaction体系. This merging of operations preserves spatial information while managing process complexity through a unified protocol rather than separate, isolated steps
4Quantity of substance
If proximity-ligated DNA molecules are fragmented to generate sequencing templates, then full genome coverage is achieved, but spatial-proximal contiguity information must be preserved through the process
Solution Approach 1:
The patent applies preliminary action by performing cross-linking before fragmentation to lock in spatial relationships. The cross-links are established in advance on intact chromatin, ensuring that when fragmentation occurs later, the spatial-proximal contiguity information is already preserved in the cross-linked structure and can be recovered through subsequent ligation and sequencing
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 ensures uniform genome coverage and accurate preservation of spatial-proximal contiguity, enhancing applications such as genomic rearrangement detection, haplotype phasing, and 3D genome interaction analysis.
Implementation Method 1
contacting cross-linked DNA molecules of a sample comprising a genome or portion thereof with a set of restriction endonucleases; thereby generating spatial-proximal digested ends of cross-linked DNA molecules
Implementation Method 2
contacting the spatial-proximal digested ends of cross-linked DNA molecules with ligase, thereby generating cross-linked proximity-ligated DNA molecules comprising ligation junctions
Implementation Method 3
contacting the cross-linked proximity-ligated DNA molecules comprising ligation junctions with a reagent that reverses cross-linking, thereby generating proximity-ligated DNA molecules comprising ligation junctions
Data Source
AI summary
Provided herein are methods and compositions for preparing sequencing templates that provide uniform genome coverage and preserve spatial-proximal contiguity information.