Single-Cell Immune Receptor Pairing via Oligonucleotide Beads
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Solution Overview
Problem
Current methods for high-throughput sequencing of immune receptor repertoires in individual cells are limited by the inability to resolve heavy and light chain pairings at high throughput, preventing the complete analysis of adaptive immune receptors.
Innovation Solution
A method involving the separation of single cells with oligonucleotide-conjugated beads, followed by mRNA hybridization, overlap extension reverse transcriptase polymerase chain reaction (RT-PCR), and sequencing to link and identify paired antibody VH and VL sequences or TCR sequences from individual cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If bulk RT-PCR is used to sequence immune receptor repertoires, then high-throughput sequencing is achieved, but heavy and light chain pairings cannot be resolved
Solution Approach 1:
The method segments the sequencing process into single-cell level analysis, where each cell's heavy and light chain transcripts are captured, reverse transcribed, and PCR amplified separately before sequencing. This segmentation enables pairing resolution while maintaining throughput by processing many cells in parallel across multiple compartments.
Solution Approach 2:
The method employs nested structures where beads containing oligonucleotides are placed within compartments, cells are lysed within these compartments, and multiple molecular operations (reverse transcription, PCR) are performed in nested steps within the same compartment system, enabling complex analysis of multiple transcripts from single cells.
2Productivity
If separate sequencing of heavy and light chains is performed, then high-throughput analysis is possible, but complete immune receptor repertoires cannot be reconstructed
Solution Approach 1:
The method merges the analysis of heavy and light chain transcripts within the same single-cell compartment system. By capturing both transcript types from individual cells and performing reverse transcription and PCR on both chains simultaneously in the same compartment, the method preserves pairing information while maintaining high-throughput capability through parallel processing of many cells.
3Measurement precision
If single-cell transcript analysis is performed, then pairing information is obtained, but throughput is limited
Solution Approach 1:
The compartment system serves multiple functions simultaneously: it isolates single cells, captures mRNA transcripts, facilitates cell lysis, enables reverse transcription, and supports PCR amplification. This multi-functionality allows accurate single-cell pairing analysis while maintaining high throughput by eliminating the need for multiple separate processing steps.
Solution Approach 2:
The method performs preliminary actions within the compartment before sequencing: cells are pre-isolated and positioned in compartments, oligonucleotide beads are pre-conjugated, and transcripts are pre-captured. This preliminary preparation enables rapid sequential processing of many cells, achieving both high accuracy in pairing detection and high throughput.
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 high-throughput sequencing of paired immune receptor chains, significantly increasing the throughput and accuracy of immune receptor repertoire analysis, allowing for the determination of complete immune repertoires in individuals or populations.
Implementation Method 1
allowing mRNA transcripts released from the cells to hybridize with oligonucleotides conjugated to the beads
Implementation Method 2
performing overlap extension reverse transcriptase polymerase chain reaction to form a single DNA molecule comprising at least two cDNAs
Implementation Method 3
performing overlap extension reverse transcriptase polymerase chain reaction to form a single DNA molecule comprising at least two cDNAs from the at least two transcripts
Data Source
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AI summary
The present disclosure generally relates to sequencing two or more genes expressed in a single cell in a high-throughput manner. More particularly, the present disclosure relates to a method for high-throughput sequencing of pairs of transcripts co-expressed in single cells (e.g., antibody VH and VL coding sequence) to determine pairs of polypeptide chains that comprise immune receptors.