Phased Read-Set Generation for Repetitive-Region Haplotype Phasing
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Solution Overview
Problem
Existing methods struggle to accurately determine haplotype phase information in complex DNA samples, such as those with diploid or polyploid genomes, due to loci being separated by repetitive regions or long stretches of identical sequence, making standard assembly insufficient for assigning phase information to alleles at a locus.
Innovation Solution
A method involving segmentally rearranged nucleic acid molecules, where DNA molecules are bound to a DNA binding moiety, cleaved to expose segments, and reattached via phosphodiester bonds to form reassembled molecules, allowing sequencing of long-distance phase information in a single read.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard assembly methods are used to sequence DNA, then the process is simple and widely applicable, but phase information cannot be accurately determined for loci separated by repetitive regions or long stretches of identical sequence
Solution Approach 1:
The DNA molecule is divided into multiple segments that are bound to the DNA binding moiety independently. These segments are then cleaved from the original phosphodiester backbone and reattached through phosphodiester bonds to form reassembled molecules. This segmentation allows segments that were previously separated by repetitive regions to be brought together in a controlled manner, enabling accurate phase information determination even for loci separated by long stretches of identical sequence.
Solution Approach 2:
A DNA binding moiety serves as an intermediary structure that temporarily holds multiple DNA segments in a controlled arrangement. This intermediary allows the segments to be positioned and oriented correctly before cleavage and reattachment, ensuring that the relative positions and phase relationships between loci are preserved during the rearrangement process.
2Loss of information
If DNA segments are bound to a DNA binding moiety and reattached via phosphodiester bonds, then phase information is preserved for segments separated by large distances, but the process requires multiple steps including binding, cleaving, and attaching
Solution Approach 1:
The DNA segments are bound to the DNA binding moiety in a controlled preliminary step before cleavage occurs. This preliminary binding arrangement ensures that when the phosphodiester backbone is cleaved and the segments are reattached, the relative positions and phase relationships are preserved. The preliminary action of binding to the moiety prevents random recombination and maintains the integrity of phase information.
Solution Approach 2:
The method changes the physical and chemical state of the DNA molecule by forming non-covalent interactions with the DNA binding moiety, then utilizing enzymatic or chemical cleavage to break phosphodiester bonds, and finally forming new phosphodiester bonds during reattachment. These parameter changes allow the DNA segments to be rearranged while preserving phase information, transforming the molecule from a continuous structure to a segmented and reassembled structure.
3Length of moving object
If segments are separated by distances greater than sequencing read length, then standard sequencing cannot read them in a single read, but the method enables sequencing of long-distance phase information
Solution Approach 1:
The method introduces a spatial reorganization dimension by binding DNA segments to a DNA binding moiety in a controlled three-dimensional arrangement. This allows segments that are far apart in the original linear sequence to be brought into close physical proximity on the moiety surface. After cleavage and reattachment, the segments are positioned such that they can be read in a single sequencing read, effectively extending the readable length beyond the physical read length limitation.
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
Preserves phase information by rearranging nucleic acid segments relative to their natural positions, enabling confident assignment of sequence segments to a common phase, even when separated by distances greater than a sequencing read length.
Implementation Method 1
contacting the first DNA molecule to a DNA binding moiety such that the first segment and the second segment are bound to the DNA binding moiety independent of a common phosphodiester backbone of the first DNA molecule
Implementation Method 2
The method often comprises contacting the first DNA molecule to a cross-linking agent, such as formaldehyde
Implementation Method 3
cleaving the first DNA molecule comprises contacting to a restriction endonuclease such as a nonspecific endonuclease
Implementation Method 4
attaching the first segment to the second segment via a phosphodiester bond to form a reassembled first DNA molecule
Data Source
AI summary
Disclosed herein are methods, compositions and systems that facilitate accurate phasing of sequence data such as genomic sequence data through the segmentation and rearrangement of nucleic acid molecules in such a way as to preserve individual molecules' phase or physical linkage information. This is variously accomplished by binding molecules independent of their phosphodiester backbones, cleaving the molecules, ligating, and sequencing the molecules through long-read sequencing technology to recover segment sequence information spanning at least more than one segment.