In Situ Cell Barcoding Using Alpha-Thiol dNTPs
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Solution Overview
Problem
Current diagnostics for genetic alterations in heterogeneous cell populations are limited by the need for physical isolation of cells and bulk tumor sequencing, which fails to provide single-cell resolution and phenotypic insights into tumor heterogeneity.
Innovation Solution
The method involves in situ combinatorial cell barcoding, where barcodes are introduced into intact cells to tag DNA or RNA fragments, eliminating the need for physical isolation and allowing for the analysis of individual cells without lysing them, using alpha-thiol modified dNTPs to protect against degradation and alpha-thiol dNTP mixes for amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If physical isolation of cells through droplet formation or cell sorting is performed, then single-cell resolution is achieved, but device complexity and processing time increase significantly
Solution Approach 1:
The patent extracts the cell isolation step entirely from the workflow. Instead of physically separating cells through droplet formation or sorting, the method works directly with intact cells in suspension, extracting only the necessary genetic material for barcoding while leaving cells intact throughout the process.
Solution Approach 2:
The patent segments the barcoding process into in situ amplification steps within intact cells, followed by a separate pooling step. This allows cells to remain intact during barcoding while still enabling combinatorial indexing through sequential amplification rounds.
2Ease of manufacture
If cell lysis is performed early in NGS library preparation, then genomic DNA extraction is enabled, but single-cell information is lost due to bulk processing
Solution Approach 1:
The patent performs in situ amplification of barcodes and library preparation steps before cell lysis. By completing these steps while cells are intact, the method preserves single-cell information throughout the process. Cell lysis occurs only after barcodes have been successfully incorporated and amplified within individual cells.
3Adaptability or versatility
If split and pool methods are used for combinatorial barcoding, then unique barcodes are assigned to each cell, but the number of processing steps and reagent volumes increase
Solution Approach 1:
The patent merges multiple split-pool operations into a single in situ amplification process. By performing combinatorial barcoding within intact cells through sequential amplification rounds, the method achieves the same unique barcode assignment capability while reducing the number of physical manipulation steps and minimizing reagent consumption.
4Quantity of substance
If bulk tumor sequencing is performed, then sufficient input material is available for sequencing, but intratumor heterogeneity and phenotypic insights are lost
Solution Approach 1:
The patent creates molecular copies of genetic material from individual cells through in situ amplification. By amplifying barcodes and library preparation materials within each intact cell before pooling, the method generates sufficient sequencing input from each single cell while preserving the unique genetic and phenotypic information of each cell type within the tumor ecosystem.
Data Source
AI summary
Aspects of the present disclosure relate generally to methods, compositions, and kits for in situ whole cell or single cell barcoding. Aspects of the present disclosure also include a computer readable-medium and a processor to carry out the steps of the method described herein. In some embodiments, the disclosure relates to whole cell or single cell barcoding performed in situ.