Combinatorial Barcoding for Linked Single-Cell Imaging and Sequencing
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Solution Overview
Problem
Current methods fail to link single cell imaging data with massively parallel next-generation sequencing data, lacking spatial information and throughput in existing protocols, and plate-based workflows do not provide efficient linked image and sequence data for single cells.
Innovation Solution
Combinatorially barcoding cells with specific binding member/oligonucleotide sub-barcodes, partitioning them to obtain image and sequence data, and linking data with shared combinatorial barcodes to generate linked image and sequence data for single cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If plate-based workflows are used to sort and analyze single cells, then cells can be processed in macro-well plates, but throughput is reduced and image data cannot be linked to NGS data
Solution Approach 1:
The patent merges imaging and sequencing operations into a single integrated workflow where cells are simultaneously subjected to both image capture and nucleic acid extraction with barcoding. This combining of operations allows both image data and sequence data to be obtained from the same cells without requiring separate plate-based sorting steps, thereby maintaining throughput while enabling data linkage through shared cell barcodes.
Solution Approach 2:
The system employs a universal barcoding approach where the same barcoded reagents used for NGS library preparation also serve as identifiers for linking image data. The barcodes are embedded in the imaging workflow itself, allowing the imaging system to simultaneously perform both imaging and sequencing identification functions, eliminating the need for separate sorting and data linkage steps.
2Measurement precision
If flow cytometry is used for single cell analysis, then cells can be sorted and analyzed, but spatial information is lost
Solution Approach 1:
The patent introduces spatial dimensionality back into single-cell analysis by performing imaging before partitioning. Cells are imaged while still in their spatial context within the flow stream, capturing positional and morphological information. After imaging, cells are partitioned into individual wells where they maintain their barcode identifiers. This dimensional approach allows simultaneous acquisition of spatial image data and high-throughput sequencing data linked by the barcodes.
3Loss of information
If single cell sorting is performed before multiomic workflows, then cells can be isolated, but image data and NGS data cannot be linked
Solution Approach 1:
The patent performs the barcoding action preliminarily during the imaging step itself, before cell partitioning and subsequent sequencing. The barcoded reagents are introduced to cells during the imaging workflow, allowing both image capture and barcode assignment to occur simultaneously. This preliminary barcoding ensures that when cells are later processed for sequencing, the barcodes are already in place to serve as linking keys between image and sequence data, eliminating the need for complex post-sorting linkage procedures.
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 efficient acquisition of spatially informative image and sequencing data for single cells, enhancing throughput and linking data from the same cells for comprehensive analysis.
Implementation Method 1
combinatorially barcoding cells, e.g., obtained from an initial cellular sample, with specific binding member/oligonucleotide sub-barcodes
Implementation Method 2
The resultant combinatorial barcoded cells are next partitioned to produce partitioned combinatorial barcoded single cells
Implementation Method 3
linking of the image data and sequence data that share a common combinatorial barcode
Data Source
AI summary
Aspects of the invention include methods of obtaining linked image and sequence data for single cells, e.g., of a cellular sample. Embodiments of the methods include: combinatorially barcoding cells, e.g., obtained from an initial cellular sample, with specific binding member/oligonucleotide sub-barcodes to produce combinatorial barcoded cells. The resultant combinatorial barcoded cells are next partitioned to produce partitioned combinatorial barcoded single cells each having a combinatorial barcode. Image data and sequence data are then obtained for the partitioned combinatorial barcoded single cells, followed by linking of the image data and sequence data that share a common combinatorial barcode in order to obtain linked image and sequence data for single cells of the cellular sample. Also provided are compositions for practicing methods of the invention.