Microfluidic DNA Enrichment to Reduce Chimeras in Long-Read Sequencing
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Solution Overview
Problem
Current methods for characterizing and verifying long-context information of DNA modifications in individual cells and cell populations, such as CRISPR-based and Xdrop-targeted enrichment, produce chimeric and branched DNA that are not optimal for long-read nanopore sequencing, leading to sequencing complexity and cost inefficiencies.
Innovation Solution
An in vitro method involving DNA fragmentation, adaptor ligation, emulsion droplet formation, specific detection, physical sorting, and long-range PCR amplification in droplets to enrich and amplify target DNA molecules, producing high-fidelity, long-range amplified DNA suitable for nanopore sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-based enrichment or Xdrop-targeted enrichment is used to characterize DNA modifications, then targeted long-read sequencing is enabled, but chimeric and branched DNA is produced that increases sequencing complexity and reduces cost efficiency
Solution Approach 1:
The method segments the DNA enrichment process into distinct functional stages: (1) DNA fragmentation into manageable sizes, (2) adaptor ligation for sequencing compatibility, (3) emulsion droplet formation for parallel processing, (4) specific detection for target identification, (5) physical sorting for enrichment, and (6) long-range PCR amplification for final preparation. This segmentation eliminates chimera formation by avoiding multi-step enrichment protocols that cause DNA structure degradation.
Solution Approach 2:
The invention extracts and removes problematic intermediate enrichment steps that generate chimeric DNA. By using a streamlined approach that goes directly from fragmentation to selective amplification via droplet sorting, the method eliminates the source of branched DNA structures that complicate sequencing analysis.
2Measurement precision
If ultra-deep sequencing is performed to provide sufficient sequence coverage per single cell genome, then complete characterization is achieved, but cost increases significantly
Solution Approach 1:
The method performs preliminary enrichment and selection of target DNA molecules before sequencing. By using emulsion droplet-based specific detection and physical sorting to pre-concentrate the desired sequences, the subsequent sequencing requires much lower depth to achieve adequate coverage, significantly reducing overall cost while maintaining precision.
Solution Approach 2:
The invention changes the parameter of DNA molecule selection from random sampling to targeted enrichment. By modifying the input to sequencing from whole genome to pre-enriched target sequences, the required sequencing depth parameter is reduced, lowering costs while maintaining or improving measurement precision for the specific targets of interest.
3Quantity of substance
If DNA molecules are fragmented and processed through multiple enrichment steps, then target enrichment is achieved, but amplification bias increases and DNA fidelity decreases
Solution Approach 1:
The method extracts and eliminates unnecessary intermediate enrichment steps that introduce amplification bias. By using a direct approach with emulsion droplet sorting followed by single-step long-range PCR, the protocol minimizes cumulative bias while maintaining high target enrichment and DNA fidelity.
Solution Approach 2:
The emulsion droplet system provides built-in feedback through specific detection of target DNA molecules within individual droplets. This allows real-time verification of enrichment success and enables selection of only those droplets containing genuine targets, reducing off-target amplification and maintaining high DNA fidelity in the final product.
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
The method results in longer, uninterrupted sequence reads with higher alignment to the reference sequence, reducing amplification bias and sequencing complexity, making it suitable for analyzing gene editing outcomes like CRISPR-Cas9 editing and CAR-T cassette integration patterns.
Implementation Method 1
forming of an emulsion of a multiple of double emulsion droplets from the liquid sample obtained in step (c)
Data Source
AI summary
Provides a method to obtain enrichment of large DNA molecules for long-read sequencing using FACS or microfluidic partitioning. Furthermore, the present invention relates to a kit comprising a plurality of 5 microfluidic devices and a plurality of fluids configured for use with the system and the method.