Ultralong DNA Target Capture for Repeat-Rich Genomic Analysis
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Solution Overview
Problem
Current genetic analysis methods, such as Sanger sequencing and short-read sequencing, are inefficient and costly, particularly when characterizing animal models, and struggle with repeat-rich regions, leading to a lack of comprehensive understanding of genetic and epigenetic alterations.
Innovation Solution
A method involving the extraction of ultralong DNA molecules, fragmentation using CRISPR/Cas9 targeting, and nanopore sequencing to determine genetic and epigenetic information, allowing for precise identification of target-specific genetic and epigenetic alterations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Sanger sequencing or PCR based assays are used to characterize animal models, then genetic information can be obtained, but the cost is very high and only about 5% of transgenic mice have their insertion site known
Solution Approach 1:
The patent changes the key parameter from short-read sequencing to ultralong-read sequencing (500kb-1Mb+), fundamentally altering the capability to span repetitive regions and accurately characterize insertion sites. This parameter change enables both high precision and high throughput by capturing complete genomic contexts in single reads.
Solution Approach 2:
The patent segments the genome into manageable ultralong fragments (500kb-1Mb) that can be sequenced individually. By dividing the genome into these segments and sequencing them with high precision, the method achieves comprehensive characterization at scale, resolving the contradiction between accuracy and throughput.
2Productivity
If standard short-read sequencing approaches are used, then sequencing can be performed, but structural data is lost and repeat-rich regions are negatively impacted
Solution Approach 1:
Instead of breaking DNA into short fragments and losing structural context, the patent inverts the approach by extracting and sequencing ultralong DNA fragments that preserve the native structural information. This inversion maintains both sequencing efficiency and structural data integrity.
Solution Approach 2:
The patent performs preliminary extraction and preservation of ultralong DNA molecules before sequencing, ensuring that structural information is maintained throughout the process. This preliminary action prevents information loss by establishing the intact structural context prior to analysis.
3Measurement precision
If ultralong DNA molecules are extracted and sequenced using nanopore sequencing, then comprehensive genetic and epigenetic information can be obtained with high resolution, but the method complexity increases
Solution Approach 1:
The nanopore sequencing platform performs multiple functions simultaneously: it sequences DNA, detects epigenetic modifications, and preserves structural information all in a single process. This multi-functionality achieves high-resolution characterization without proportionally increasing complexity, as one device handles multiple analytical tasks.
4Measurement precision
If CRISPR/Cas9 targeted cleavage is used to fragment ultralong DNA molecules, then specific genomic regions can be targeted, but the method requires multiple steps including adapter ligation
Solution Approach 1:
The patent uses CRISPR/Cas9 as an intermediary to bridge specific genomic targeting and ultralong-read sequencing. The Cas9-gRNA complex acts as a mediator that precisely locates and cleaves target regions, enabling specific genomic analysis while maintaining the benefits of ultralong-read technology.
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
Enables precise and comprehensive characterization of genomic regions, including repeat-rich areas, with high resolution and cost-effectiveness, facilitating the assessment of genetic modifications and epigenetic changes.
Implementation Method 1
measuring an ionic current when a single-stranded DNA fragment of the extracted ultralong DNA molecule exposed to a voltage passes through a nanopore
Implementation Method 2
contacting the bound preselected Cas9 sgRNAs with a plurality of one or more Cas9 enzymes; cutting the ultralong DNA molecule at the Cas9/sgRNA complexes
Data Source
Figure 1A
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AI summary
The invention, in some aspects, relates to methods and systems comprising long-read sequencing of DNA molecules for identifying target-specific genetic and epigenetic alterations in DNA sequence of interest.