Single-Cell Chromatin Accessibility Genotyping at Targeted Loci
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods struggle to accurately connect genotypes to cellular phenotypes in primary human samples due to the admixture of normal and mutant cells and high cellular heterogeneity, particularly in studying clonal expansions in healthy and malignant tissues.
Innovation Solution
A method involving generating biological particles with genomic DNA and chromatin, fragmenting them using a transposase, and employing primer pairs and barcoded beads to amplify and sequence locus nucleic acid fragments, providing chromatin accessibility and genotyping information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bulk tissue analysis is performed, then statistical power is improved, but cellular heterogeneity is lost
Solution Approach 1:
The bulk tissue sample is segmented into individual single cells through enzymatic dissociation and filtration processes, allowing analysis at cellular resolution while maintaining statistical power through high cell numbers
2Loss of information
If single-cell analysis is performed, then cellular heterogeneity is preserved, but statistical power deteriorates
Solution Approach 1:
Genomic DNA is amplified using multiple displacement amplification (MDA) to create sufficient copies from single-cell quantities, enabling downstream genotyping applications with adequate signal strength
Solution Approach 2:
The assay design uses targeted loci with high allele frequency differences and optimized probe concentrations to enhance signal detection sensitivity at single-cell levels
3Area of stationary object
If whole-genome amplification is performed, then genomic coverage is improved, but allele dropout increases
Solution Approach 1:
Instead of amplifying the entire genome, the method extracts and amplifies only specific targeted loci, reducing amplification bias and allele dropout while maintaining sufficient coverage for genotyping
Solution Approach 2:
The assay optimizes amplification conditions specifically for targeted loci with high allele frequency differences, rather than attempting uniform whole-genome amplification
4Measurement precision
If targeted loci with high allele frequency differences are used, then genotyping accuracy is improved, but locus selection complexity increases
Solution Approach 1:
The assay design selects targeted loci that serve multiple purposes: they provide high genotyping accuracy through allele frequency differences, work across diverse cell types, and are compatible with the single-cell amplification methodology
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 accurate single-cell genotyping and chromatin accessibility mapping, improving the ability to resolve cellular complexity and connect genotypes to phenotypes, especially in clonal mosaicism.
Implementation Method 1
The cell permeabilization solution (CPS) of the present disclosure facilitates chromatin release from the cell nucleus
Implementation Method 2
The Tn5-based single-cell chromatin accessibility assay (scATAC-seq) has emerged as a powerful tool for profiling chromatin accessibility at single-cell resolution
Implementation Method 3
magnetic bead-based single-cell isolation method
Implementation Method 4
single-cell whole-genome amplification using multiple displacement amplification (MDA)
Data Source
Figure 1a
Figure 1b~1c
Figure 1d~1e
AI summary
The present disclosure relates generally to methods, compositions, and systems for processing nucleic acids from individual cells or cell populations. The present invention enables direct sequencing of full-length RNA molecules with no amplification steps. The present invention allows determination of RNA modifications in highly specific cell populations of any tissue.