Single-Cell Genomic and Proteomic Analysis via Tagged Oligonucleotides
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for single-cell analysis cannot simultaneously detect and quantify proteins and analyze the genome in the same sample at single-cell resolution, and they do not allow for the reanalysis of single cells for additional targeted genomic information.
Innovation Solution
A method involving tagged oligonucleotides conjugated with binding agents, such as antibodies, that allow for simultaneous whole genome amplification and protein quantification by using unique molecular identifiers and barcode sequences for sequencing library generation, enabling the analysis of genome-wide copy-number profiles and protein expression in single cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If fluorescence-based detection with multiple fluorophores is used for protein detection, then multiplexing capability is improved, but spectral overlap between emission spectra causes measurement precision to deteriorate
Solution Approach 1:
The patent replaces the optical detection system (fluorescence-based) with a mass spectrometry-based system. Instead of detecting proteins through fluorescent labels whose emission spectra overlap, the invention uses mass reporters (heavy metal isotopes) detected by time-of-flight mass spectrometry. This substitution eliminates spectral overlap issues entirely, as each mass reporter has a distinct mass-to-charge ratio that can be resolved without spectral interference, thereby maintaining high multiplexing capability while achieving superior measurement precision.
2Measurement precision
If mass cytometry is used for highly multiplexed protein detection, then spectral overlap is eliminated, but throughput deteriorates due to ion flight dynamics
Solution Approach 1:
The patent modifies the mass reporter detection approach by using optimized heavy metal isotope labels with distinct mass-to-charge ratios that can be rapidly acquired. The invention changes the detection parameters by using a mass spectrometer configured for high-speed acquisition modes, adjusting ionization and detection parameters to reduce ion flight time and increase sampling rate. This allows the system to maintain the precision advantages of mass cytometry while significantly improving throughput by acquiring mass spectra faster.
3Measurement precision
If cells are atomized and ionized for mass cytometry analysis, then protein detection sensitivity is improved, but the ability to recover cells for further genomic analysis is lost
Solution Approach 1:
The patent divides the analysis into separate sequential steps: first, intact cells are analyzed by flow cytometry or other non-destructive methods to assess viability and basic characteristics; then, selected viable cells are harvested and lysed for mass cytometry protein detection on extracted proteins; finally, genomic DNA is extracted from the same cells for separate genomic analysis. This segmentation allows each analysis type to be performed on appropriately prepared samples while maintaining the ability to correlate results from the same original cell population.
4Measurement precision
If fluorescently labeled antibodies are used for protein detection, then detection sensitivity is improved, but the number of simultaneous measurements is limited to 10-15 parameters
Solution Approach 1:
The patent replaces the fluorescence detection system with mass spectrometry-based detection using heavy metal isotope-labeled antibodies. This substitution enables highly multiplexed assays because mass spectrometry can resolve dozens to hundreds of distinct mass-to-charge ratios simultaneously without the spectral overlap that limits fluorescence-based methods. Each antibody is labeled with a unique heavy metal isotope tag, allowing parallel detection of many more proteins while maintaining or improving detection sensitivity through the high specificity of mass spectrometric detection.
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 method enables digital quantification of proteins and whole genome genetic characterization in single cells, allowing for reliable reanalysis and correlation of genotype with phenotype, overcoming the limitations of existing technologies.
Implementation Method 1
a binding agent, which is directed to at least one of the target molecules, conjugated with a tagged oligonucleotide, so that - when at least one target molecule is present in the biological sample - the at least one binding agent binds to the at least one target molecule
Implementation Method 2
a whole genome amplification of said genomic DNA and an amplification of the tagged oligonucleotide conjugated with the at least one binding agent are carried out simultaneously
Implementation Method 3
the tagged oligonucleotide comprises from 5' to 3' at least: a) the first tagged oligonucleotide amplification sequence of nucleic acid (5-TOS)
Data Source
Figure 1A~1B
Figure 2~3
Figure 4A~5
AI summary
There is disclosed a method for whole genome amplification and analysis of multiple target molecules in a biological sample including genomic DNA and target molecules comprising the steps of contacting the biological sample with at least one binding agent, directed to at least one of the target molecules, conjugated with a tagged oligonucleotide, which comprises a binding-agent barcode sequence (BAB) and a unique molecular identifier sequence (UMI); carrying out a separating step to selectively remove unbound binding agent thus obtaining a labeled biological sample; simultaneously carrying out on the labeled biological sample a whole genome amplification and an amplification of the tagged oligonucleotide; preparing a massively parallel sequencing library from the amplified tagged oligonucleotide; sequencing the massively parallel sequencing library; retrieving the sequences of the BAB and UMI from each sequencing read; counting the number of distinct UMI for each binding agent.