Single-Cell RNA Sequencing With Pooled Barcodes for Rare Transcripts
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Solution Overview
Problem
Current single-cell RNA sequencing methods face challenges such as reduced throughput, batch effects, and high costs, particularly in well-based methods, while microfluidics-based methods require fresh samples and suffer from batch effects when sequencing at different time points.
Innovation Solution
A method involving reverse transcription, template switching, pooling, amplification, and tagmentation, using gene-specific primers and cell barcodes, enables high-throughput, cost-effective, and sensitive analysis of single-cell transcriptomes, allowing for the enrichment and quantification of rare sequences alongside whole transcriptomic analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If well-based sequencing methods are used, then sample storage and reduced batch effects are improved, but throughput and processing efficiency deteriorate
Solution Approach 1:
The patent combines the advantages of well-based methods (sample storage capability, reduced batch effects) with microfluidics-based methods (high throughput) by implementing a hybrid approach where cells are sorted into wells, lysed, and reverse transcribed individually, then pooled and processed together through amplification and sequencing, achieving both reliability and productivity
2Productivity
If microfluidics-based methods are used, then throughput and ease of operation are improved, but sample flexibility and batch effect control worsen
Solution Approach 1:
The patent segments the sequencing process into independent well-based reactions where each well can process individual cells or small groups of cells from different samples and time points, allowing flexible sample accumulation before pooled processing, thus maintaining high throughput while improving sample flexibility and reducing batch effects
3Productivity
If PCR amplification is used, then amplification efficiency is improved, but product loss and sensitivity to degradation worsen
Solution Approach 1:
The patent replaces PCR amplification (enzymatic/mechanical system) with in vitro transcription (IVT) amplification, which uses T7 RNA polymerase to amplify cDNA. This substitution reduces product loss and degradation sensitivity because IVT produces RNA products that are more stable and less prone to degradation during sample handling, while maintaining sufficient amplification efficiency
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
The method provides comprehensive and sensitive sequencing data with reduced batch effects, enabling accurate detection and quantification of rare genes, and allows for pooling of libraries, thus increasing throughput and reducing costs.
Implementation Method 1
reverse transcription, template switching, pooling, amplification, and tagmentation
Implementation Method 2
amplification step, which in 10× and SS2 is done via PCR amplification
Data Source
AI summary
The present invention provides methods and compositions for analysis multiplexed RNA transcriptomes at the single cell level.