Preserving Spatial-Proximal Contiguity in Nucleic Acid Templates
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Solution Overview
Problem
Next-generation sequencing (NGS) methods face limitations in preserving spatial-proximal and molecular contiguity of nucleic acid templates, leading to suboptimal sequencing data quality, particularly in determining genomic variants and haplotype phasing.
Innovation Solution
The method CPSP-Prep involves capturing spatially proximal nucleic acid molecules using proximity ligation or solid substrate-mediated proximity capture, followed by compartmentalization and tagging with molecular barcodes to preserve both spatial-proximal and molecular contiguity, enabling the generation of contiguity-preserved nucleic acid templates suitable for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If nucleic acids are fragmented for NGS, then sequencing compatibility is improved, but spatial-proximal and molecular contiguity are lost
Solution Approach 1:
The method segments the nucleic acid processing into distinct stages: first capturing spatial-proximal relationships through proximity ligation to create ligated products, then fragmenting only these ligated products for sequencing. This segmentation allows preservation of contiguity information in the ligated products while enabling sequencing of fragmented templates.
Solution Approach 2:
The method performs preliminary proximity ligation and capture of spatially proximal nucleic acid molecules before fragmentation. This preliminary action establishes contiguity relationships that are then preserved through subsequent processing steps, allowing the fragmented sequencing templates to retain information about their original spatial relationships.
2Loss of information
If proximity ligation is performed to capture spatially proximal nucleic acid molecules, then spatial-proximal contiguity is preserved, but molecular contiguity is disrupted
Solution Approach 1:
The method merges two forms of contiguity preservation: spatial-proximal contiguity through proximity ligation and molecular contiguity through careful handling of the ligated products. By merging these approaches and fragmenting only the ligated products rather than the original high molecular weight nucleic acids, both forms of contiguity are preserved in the sequencing data.
Solution Approach 2:
The method applies different quality preservation strategies to different aspects of the nucleic acid: spatial-proximal relationships are preserved through proximity ligation at specific locations, while molecular contiguity is preserved by maintaining the integrity of the ligated product molecules until the fragmentation step. Each aspect receives localized preservation treatment appropriate to its nature.
3Stability of the object's composition
If high molecular weight nucleic acids are used, then molecular contiguity is preserved, but spatial-proximal capture efficiency decreases
Solution Approach 1:
The method performs preliminary fragmentation of the high molecular weight nucleic acids to create substrates of appropriate size for efficient proximity ligation and capture. This preliminary action increases the surface area and accessibility of nucleic acid molecules, thereby improving spatial-proximal capture efficiency while the subsequent ligation and careful handling preserve molecular contiguity information.
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
CPSP-Prep enhances the determination of nucleic acid sequences by improving variant sensitivity and haplotype phasing, allowing for more accurate genome assembly and identification of genomic variants, while also preserving conformation and topology information.
Implementation Method 1
nSPNAs are captured by ligation to generate ligated products (LP)
Implementation Method 2
solid substrate-mediated proximity capture
Data Source
AI summary
Provided herein are methods and compositions for preparing nucleic acid templates wherein spatial-proximal and molecular contiguity of target nucleic acids is preserved, and the sequencing data obtained therefrom is used, but not limited to, identification of genomic variants, determination of contiguity information to inform assemblies of target nucleic acids de novo including deconvolution of haplotype phase information, and analyses of conformation and topology of target nucleic acids.