Long Insert Whole Genome Sequencing for Structural Variant Detection
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Solution Overview
Problem
Current whole genome sequencing methods require significant DNA input and are not suitable for analyzing smaller DNA samples, such as those from tumor biopsies, and often require post-sequencing trimming due to enzymatic footprints, making them inefficient for detecting genomic rearrangements like copy number variants and translocations.
Innovation Solution
A method involving Long Insert Whole Genome Sequencing (LI-WGS) that fragments DNA to 800-1,100 base pairs, purifies and amplifies the inserts using magnetic beads, and sequences them to detect genomic rearrangements without the need for circularization or biotin pull-down, allowing for increased physical coverage and analysis of larger regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mate pair library preparation protocol is used to evaluate larger regions, then the ability to detect structural variants improves, but the DNA input requirement increases to 10 μg which is not available from tumor biopsv
Solution Approach 1:
The patent changes the insert size parameter from standard short insert (300-500 bp) to long insert (800-1,100 bp) while maintaining the same library preparation workflow. This parameter change enables detection of larger structural variants and copy number changes without requiring increased DNA input, as the long inserts provide better span across rearrangement breakpoints while being compatible with low input DNA (1-10 ng) whole genome sequencing
2Quantity of substance
If Nextera Mate Pair Sample Preparation Kit is used, then DNA input requirement decreases to 1-4 μg, but enzymatic footprint from transposome-mediated fragmentation requires post-sequencing trimming
Solution Approach 1:
The patent replaces enzymatic fragmentation (transposome-mediated) with mechanical sonication-based fragmentation. This substitution eliminates the enzymatic footprint that requires post-sequencing trimming, as sonication creates clean breaks without enzymatic modification artifacts. The mechanical fragmentation method maintains compatibility with low DNA input requirements while producing cleaner sequencing data that requires no additional trimming steps
3Loss of energy
If standard whole genome sequencing is performed, then cost decreases compared to exome sequencing, but physical coverage of larger regions is insufficient for detecting breakpoints
Solution Approach 1:
The patent changes the insert size parameter from standard short insert to long insert (800-1,100 bp) in whole genome sequencing. This parameter change increases physical coverage of genomic regions between reads, enabling detection of breakpoints and structural variants while maintaining cost effectiveness. The long inserts provide better span across rearrangement events without requiring additional sequencing depth or more expensive targeted approaches
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
LI-WGS increases the power to detect copy number variants and translocations by achieving higher physical coverage with less sequencing, reducing the need for DNA input and eliminating post-sequencing trimming steps, making it a more cost-effective and user-friendly approach compared to traditional methods.
Implementation Method 1
fragmenting the nucleic acid sample with sonication to produce a plurality of inserts with a length of about 800 to 1,100 base pairs
Implementation Method 2
purifying the plurality of inserts using magnetic beads
Data Source
AI summary
The present invention is directed to a method of detecting a genomic rearrangement in a nucleic acid sample with Long Insert Whole Genome Sequencing (LI-WGS). The method may include obtaining a nucleic acid sample and then fragmenting the nucleic acid sample (e.g., via sonication). In particular, the fragmenting may result in the production of a plurality of inserts. Thereafter, the method comprises purifying the plurality of inserts using magnetic beads and then amplifying the purified plurality of inserts. In addition, the method further comprises sequencing the purified and amplified plurality of inserts. In some aspects, the plurality of inserts have a length of between about 800 and about 1,100 base pairs.