In Situ Library Preparation for Rare Cell Sequencing
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Solution Overview
Problem
Current target enrichment methods for genomic alterations, such as multiplexed PCR and hybrid capture, struggle with scaling to small cell populations and lose spatial and phenotypic information due to cell lysis, making them costly and inefficient for analyzing rare cell populations like cancer cells and tumor-infiltrating immune cells.
Innovation Solution
In situ amplicon-based and ligation-based library preparation methods that maintain cells intact, allowing for the analysis of small cell populations by performing library preparation within individual cells, preserving phenotypic markers and enabling the enrichment of rare cell populations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional library preparation methods are used with purified genomic DNA from large cell populations, then sequencing coverage is sufficient, but spatial information and phenotypic information of rare cell populations are lost
Solution Approach 1:
The method segments the library preparation process by performing it individually within each cell using cell-permeable beads delivered via microinjection or electroporation. Each cell serves as an isolated reaction vessel, preventing cross-contamination and preserving spatial information while enabling rare cell analysis without requiring large population enrichment
Solution Approach 2:
The invention nests the library preparation reaction inside individual cells by introducing cell-permeable beads containing fragmentation enzymes, adapters, and barcodes that enter cells and perform in-situ DNA processing. This nested approach allows sequencing library preparation to occur within the natural confines of each cell, preserving phenotypic information
2Ease of manufacture
If cells are lysed before library preparation, then DNA extraction is efficient, but phenotypic markers and spatial context are lost
Solution Approach 1:
The method performs preliminary library preparation steps (fragmentation, adapter ligation, barcode incorporation) inside intact cells before lysis and DNA extraction. This preliminary action within living cells preserves phenotypic markers and spatial context that would otherwise be lost during traditional lysis-based extraction protocols
Solution Approach 2:
Cell-permeable beads act as intermediaries that deliver fragmentation enzymes, adapters, and barcodes into intact cells without requiring cell lysis. These beads mediate the library preparation process within the cellular environment, enabling DNA processing while preserving cell integrity and associated phenotypic information
3Measurement precision
If multiple sorting steps are performed to enrich rare cell populations, then detection sensitivity improves, but cost and complexity increase
Solution Approach 1:
Each cell performs self-service library preparation through the introduced cell-permeable beads that contain all necessary reagents (fragmentation enzymes, adapters, barcodes). This self-contained approach eliminates the need for complex multi-step sorting and enrichment procedures, reducing operational complexity while maintaining detection sensitivity for rare cell populations
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 the high-yield enrichment and sequencing of subpopulations of 10 cells or less, preserving phenotypic information and providing detailed insights into tumor ecosystems, improving the detection sensitivity of disease-associated genetic alterations in heterogeneous cell populations.
Implementation Method 1
the method utilizes the cell membrane to contain the genetic information into individual cell reactions within a single reaction
Data Source
AI summary
Aspects of the present disclosure relate generally to methods, compositions, and kits for preparing a ligation-based or amplicon-based library in situ for sequencing.