Target-Capture Ultralong-Read Sequencing for Transgene Mapping
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Solution Overview
Problem
Current genetic analysis methods, such as Sanger sequencing and PCR-based assays, are costly and inefficient for characterizing animal models, particularly in regions with repeats, leading to loss of structural data and limited knowledge of transgenic mouse insertion sites, while short-read sequencing fails to capture structural variations effectively.
Innovation Solution
A method involving extraction of ultralong DNA molecules, fragmentation, adaptor ligation, and nanopore sequencing to determine genetic and epigenetic information, with optional CRISPR/Cas9 targeting for precise cleavage and sequencing adaptors, enabling identification of genetic and epigenetic alterations in target-specific genomic regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Sanger sequencing or PCR based assays are used for mouse model validation, then genetic analysis can be performed, but the cost increases and only a small percentage of transgenic mice have known insertion sites
Solution Approach 1:
The patent uses ultralong DNA molecules as intermediaries that simultaneously carry both the transgene and flanking host genomic sequences. This mediator enables single-molecule sequencing to reveal insertion sites without requiring separate validation assays for each mouse model, thereby increasing throughput while maintaining precision.
Solution Approach 2:
The patent segments the genomic DNA into ultralong molecules that span the transgene insertion site, allowing simultaneous analysis of both the transgene and host genome in a single sequencing reaction. This segmentation strategy enables comprehensive characterization of multiple mice through pooled sequencing.
2Productivity
If 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:
The patent transitions from short-read to ultralong-read sequencing, adding the dimension of extended read length. This dimensional change enables continuous sequencing through repeat-rich regions and capture of structural variations that would be fragmented or lost in short-read approaches, while maintaining sequencing efficiency through targeted enrichment.
3Measurement precision
If conventional sequencing methods are used for transgenic mouse analysis, then genetic information can be obtained, but the cost becomes very high
Solution Approach 1:
The patent creates a universal sequencing approach using ultralong DNA molecules that can characterize multiple transgenic mice simultaneously through pooled sequencing. This multi-functional method replaces the need for separate Sanger sequencing or PCR assays for each mouse, dramatically reducing the total cost while maintaining comprehensive insertion site characterization.
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
Provides precise and comprehensive characterization of chromosomal regions with high resolution and cost-efficiency, allowing for detailed analysis of genetic and epigenetic alterations, including transgene integration and off-target effects, in various biological samples.
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; wherein the preselected Cas9 sgRNAs bound to the extracted ultralong DNA molecule each bind a Cas9 enzyme, forming a plurality of Cas9/sgRNA complex sites
Implementation Method 3
cutting the ultralong DNA molecule at the Cas9/sgRNA complexes thereby producing the DNA molecule fragments
Data Source
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.