Ultra-high-throughput Single Cell Sequencing via Dual Barcode Segmentation
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Solution Overview
Problem
Current single cell sequencing technologies face limitations in throughput, leading to inefficiencies and high costs due to issues like cell contamination, low reaction efficiency, and batch effects, which result in the loss of biological information and increased sequencing costs.
Innovation Solution
An ultra-high-throughput single cell sequencing method using molecular labeled microbeads with a dual cell barcode system and a specific molecular barcode transposase-embedded complex, allowing for the distinction of multiple cells bound to the same microbead through a combination of cell barcode sequences and transposase-accessible chromatin sequencing, thereby improving sequencing efficiency and reducing contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the capture rate of cells in the microwell plate is increased to improve throughput, then more cells can be sequenced per run, but two or more cells will be captured in one microwell causing contamination of transcripts between cells
Solution Approach 1:
The patent divides the cell barcode sequence into two parts: a first cell barcode sequence on the microbead and a second cell barcode sequence in the reverse transcription sequence. This segmentation allows the system to distinguish between multiple cells bound to the same microbead by combining both barcode sequences, thereby maintaining high throughput while preventing contamination between cells
Solution Approach 2:
The patent introduces a bridge sequence as an intermediary element that connects the first cell barcode sequence on the microbead with the second cell barcode sequence in the reverse transcription sequence. This bridge sequence enables the ligation reaction between the microbead and the reverse transcription sequence, allowing both barcode sequences to be combined and used for cell identification, thus resolving the contradiction between throughput and contamination
2Reliability
If the capture rate of cells is reduced to approximately 1/10 of the total number of wells to avoid cell contamination, then each bead captures only one cell, but a large number of droplets or microwells have empty microbeads without cells, greatly reducing experimental efficiency and increasing cost
Solution Approach 1:
The patent segments the cell identification into two parts: microbead-based first cell barcode sequences and reverse transcription sequence-based second cell barcode sequences. This segmentation enables the system to accurately identify cells even when multiple cells are bound to the same microbead, allowing higher capture rates while maintaining single-cell resolution and improving experimental efficiency
Solution Approach 2:
The patent adds a second dimension of cell identification by incorporating the second cell barcode sequence in the reverse transcription sequence. This dimensional expansion allows the system to distinguish cells based on multiple sequences rather than relying solely on spatial separation in microwells, thereby enabling higher capture rates without sacrificing single-cell accuracy
3Productivity
If intracellular ligation reactions are used to improve throughput as in sci-RNA-seq and SPLiT-seq, then throughput per run is greatly improved, but reaction efficiency is low and contamination rate is high due to easy leakage of transcripts between cells
Solution Approach 1:
The patent uses a bridge sequence as an intermediary to connect the microbead with the reverse transcription sequence through ligation. This bridge sequence acts as a stable connector that prevents transcript leakage between cells while enabling the ligation reaction to proceed efficiently, thus resolving the contradiction between high throughput and low reaction efficiency
Solution Approach 2:
The patent replaces the traditional intracellular ligation reaction mechanism with a microbead-based extracellular ligation system. By using molecular labeled microbeads that capture cells and enable ligation outside the cell, the system achieves high throughput while improving reaction efficiency and reducing contamination compared to intracellular ligation methods
4Productivity
If combinatorial microfluidics is used to increase throughput as in Paul et al. (2019), then throughput is increased up to 15-fold, but cells are not sequenced in parallel leading to significant batch effect with abnormal UMI/Gene ratios, and equipment is expensive and difficult to carry
Solution Approach 1:
The patent segments the sequencing process into two independent parts: microbead-based capture and reverse transcription sequence-based labeling. This segmentation allows cells to be sequenced in parallel while maintaining simple equipment requirements, as the method can be performed using standard microfluidics or microwell plate technology without complex combinatorial microfluidics systems
Solution Approach 2:
The patent uses molecular barcodes as copies of cell identity information that can be read through sequencing. By using sequence-based identification rather than physical manipulation of individual cells, the system achieves high throughput with simpler equipment that is easier to carry and operate, avoiding the need for expensive combinatorial microfluidics machinery
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 the sequencing of millions of single cells per run, significantly enhancing throughput and reducing costs by minimizing contamination and batch effects, while maintaining high capture rates of cells and microbeads.
Implementation Method 1
ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) employs a modified Tn5 transposase that randomly inserts a specific DNA sequence as a transposon into an open chromatin region
Implementation Method 2
a poly-T tail for complementary pairing with intracellular mRNA with a poly-A sequence
Implementation Method 3
ligating the first bridge sequence with the second bridge sequence by the pairing of bridge primers with the first bridge sequence and the second bridge sequence, respectively, followed by ligation through a ligase
Data Source
AI summary
The present invention discloses an ultra-high-throughput single cell sequencing method, the method of the present invention including: firstly performing intracellular reverse transcription by using a reverse transcription sequence, or firstly performing intranuclear transposition of a transposase-accessible chromatin genome sequence by using a specific molecular barcode transposase-embedded complex, then compartmentalizing one or more cells or nuclei with one molecular labeled microbead by microwell-plate technology or microfluidic technology, followed by lysis of the cells or nuclei under the action of a lysis buffer, ligation of the sequences with the molecular label sequence on the molecular labeled microbeads through bridge primers, and PCR amplification to obtain a large quantity of sequences for construction of cDNA sequencing library, and then performing high-throughput sequencing, where information of specific transcriptome/genome accessibility of millions of single cells can be obtained in one run of sequencing. The throughput of single cell sequencing is highly improved.