Barcoded Capture Objects for Single-Cell Phenotype Linkage
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Solution Overview
Problem
Current methods struggle to link genomic and transcriptomic sequence data with the specific phenotype of individual biological cells, especially in a microfluidic environment.
Innovation Solution
The use of barcoded capture objects within a microfluidic device allows for the generation of barcoded RNA-seq and genomic DNA libraries from single cells or small clonal populations, enabling the linkage of sequence data with the cell of origin and its phenotype by reading the barcode both in situ and from sequencing data.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single cell genome amplification and next generation sequencing methods are used, then the ability to sequence genome and transcriptome of individual cells is improved, but the ability to link sequence data with specific cell phenotype is lost
Solution Approach 1:
The barcode sequence is divided into multiple cassetable oligonucleotide sequences (sub-words) that can be independently decoded. This segmentation allows the barcode to be read both in situ within the microfluidic device and from sequencing data, thereby maintaining the link between sequence data and cell phenotype while enabling high-precision sequencing
Solution Approach 2:
A barcode sequence comprising cassetable oligonucleotide sequences serves as an intermediary element that bridges the cell phenotype information and the sequencing data. The barcode is attached to capture objects that interact with individual cells, allowing phenotype information to be carried through the sequencing process and recovered in the sequence reads
2Loss of information
If barcoded capture objects with cassetable oligonucleotide sequences are used, then the ability to read barcode in situ and from sequencing data is improved, but the complexity of designing and decoding barcodes increases
Solution Approach 1:
The set of hybridization probes is designed to universally decode any combination of cassetable oligonucleotide sequences in the barcode. Each probe targets a specific sub-word sequence and can be used to decode any position in the barcode where that sub-word appears, making the decoding system versatile and reducing the need for sequence-specific decoding mechanisms
Solution Approach 2:
Instead of designing completely unique sequences for each barcode variant, the system uses a limited set of cassetable oligonucleotide sequences that are combined in different arrangements to generate diverse barcodes. This partial action approach reduces design complexity while still providing sufficient barcode diversity through combinatorial arrangements of the cassetable elements
3Loss of information
If cells are selectively disposed within sequestration pens for phenotype observation, then the linkage of genomic data with cell phenotype is improved, but the device complexity and processing time increase
Solution Approach 1:
The microfluidic device employs a nested structure with an enclosure containing multiple sequestration pens, each capable of holding individual cells or small cell populations. This nested organization allows systematic processing of multiple cells while maintaining the ability to link each cell's phenotype with its genomic data through the barcode system
Solution Approach 2:
Cells are selectively disposed within sequestration pens and their phenotypes are observed before being processed for genome and transcriptome sequencing. This preliminary action of phenotype characterization before sequencing ensures that phenotype information is captured and can be linked to the subsequent sequencing data through the barcode attached to capture objects
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
This approach enables the correlation of genomic and transcriptomic data with the observed phenotype of individual cells, providing a powerful tool for understanding cellular biology and function.
Implementation Method 1
identifying/decoding the barcode sequence of the capture oligonucleotides of the capture object in situ, using a set of complementary hybridization probes
Data Source
AI summary
Apparatuses, compositions and processes for DNA barcode deconvolution are described herein. A DNA barcode may be used to provide a bead specific identifier, which may be detected in situ using hybridization strategies. The DNA barcode provides identification by sequencing analysis. The dual mode of detection may be used in a wide variety of applications to link positional information with assay information including but not limited to genetic analysis. Methods are described for generation of barcoded single cell sequencing libraries. Isolation of nucleic acids from a single cell within a microfluidic environment can provide the foundation for cell specific sequencing library preparation.