Multiplexed Single Cell Gene Expression via Template Switch Tagmentation
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Solution Overview
Problem
Current methods for gene expression profiling, such as microarray hybridization, are limited in analyzing alternative splicing, promoters, and polyadenylation signals, and fail to capture the functional information present in single cells, leading to loss of data on dynamic processes and distinct cell types.
Innovation Solution
The method involves preparing cDNA libraries from single cells using droplets or beads with unique barcodes, such as unique molecular identifiers (UMIs), to enable multiplexed single cell gene expression analysis by releasing mRNA, synthesizing tagged cDNA, and performing tagmentation reactions to generate sequencing-ready fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If bulk mRNA analysis is performed, then processing efficiency is improved, but single-cell functional information is lost
Solution Approach 1:
The patent segments the bulk mRNA sample into individual single-cell mRNA samples, allowing each cell's transcriptome to be analyzed separately. This is achieved by isolating individual cells and performing reverse transcription on each cell's mRNA independently, thereby preserving single-cell functional information while maintaining high-throughput processing capabilities through parallelization.
Solution Approach 2:
The patent combines multiple single-cell cDNA samples into a single pooled library for sequencing. After individual cell processing and barcoding, the cDNA products from many cells are merged into one library, enabling simultaneous analysis of numerous single cells in a high-throughput manner that maintains both single-cell resolution and processing efficiency.
2Measurement precision
If microarray hybridization is used, then known genes can be analyzed, but alternative splicing, promoters and polyadenylation signals cannot be detected
Solution Approach 1:
The patent employs a universal reverse transcription and sequencing approach that can detect all types of transcript features including known genes, alternative splicing variants, promoters, and polyadenylation signals. The method uses universal primers and a unified workflow that adapts to various transcript types, providing multi-functional analysis capability beyond the limitations of microarray hybridization.
Solution Approach 2:
The patent changes the detection parameter from hybridization-based (microarray) to sequencing-based, enabling detection of diverse transcript features. By using next-generation sequencing technology with appropriate library preparation, the method can identify various transcript types and modifications that were invisible to microarray technology, thereby expanding analysis versatility.
3Measurement precision
If single cells are isolated for study, then distinct cell types can be analyzed, but insufficient cell numbers reduce statistical power
Solution Approach 1:
The patent combines cDNA products from many individually processed single cells into a pooled library, accumulating sufficient molecular material for robust statistical analysis. This merging approach maintains the resolution benefits of single-cell analysis while achieving the quantity needed for powerful statistical inference across cell types and states.
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 allows for high-throughput, cost-effective analysis of gene expression in single cells, enabling the detection of a significant number of genes and providing comprehensive transcript coverage, particularly at the 3' end of transcripts, while minimizing concatenation byproducts.
Implementation Method 1
synthesizing a first strand of cDNA from the mRNA in each individual mRNA sample with a first strand synthesis primer
Implementation Method 2
The TSO primer is extended by the reverse transcriptase when the reverse transcriptase reaches the 3' end of the first strand cDNA
Implementation Method 3
performing a tagmentation reaction to simultaneously cleave each cDNA and incorporate an adapter into each strand of the cDNA
Data Source
AI summary
Presented herein are methods and compositions for multiplexed single cell gene expression analysis. Some methods and compositions include the use of droplets and/or beads bearing unique barcodes such as unique molecular barcodes (UMI).