Single-Cell Lymphocyte Receptor Pairing in High-Throughput Sequencing
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Solution Overview
Problem
Current high-throughput sequencing technologies for antibody and T-cell receptor sequences fail to provide information on correct chain-pairing of lymphocyte receptor chains, leading to loss of critical pairing information when sequencing is performed on large numbers of cells, making it difficult to reconstitute the immune system.
Innovation Solution
A method involving sample partitioning, expansion, and sequencing of lymphocyte receptor chains in individual vessels, followed by statistical analysis to determine independent occurrences and identify functional lymphocyte receptor chain pairs, using techniques such as microfluidic chambers and next-generation sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high-throughput sequencing is performed on large numbers of cells, then sequencing productivity is improved, but pairing information is lost
Solution Approach 1:
The method segments the lymphocyte population into individual cells, each assigned to a separate reaction vessel or well. This segmentation preserves the association between heavy and light chain sequences from the same cell while enabling parallel processing of many cells simultaneously, thus maintaining pairing information at scale
Solution Approach 2:
The patent introduces barcodes or molecular identifiers as intermediaries that link heavy and light chain sequences to their cell of origin. These intermediaries enable the reconstruction of chain pairs from sequenced data by matching barcodes, thus preserving pairing information through the sequencing process
2Productivity
If sequencing is performed on bulk cell populations, then processing efficiency is improved, but measurement precision of chain pairing is degraded
Solution Approach 1:
By partitioning the bulk cell population into individually processed units (wells or reaction vessels), the method achieves both high throughput and single-cell resolution. Each segmented unit maintains complete pairing information while the collective processing of many segments delivers high productivity
Solution Approach 2:
The method creates copies of cellular material (DNA, RNA) from individual cells for sequencing, while the barcode copy maintains the link to the original cell. This copying approach enables amplification and sequencing without losing the pairing information present in the original single-cell context
Data Source
AI summary
Provided herein are high-throughput sequencing methods to study the diversity and functionality of lymphocyte receptor chains and pairing of the same. Specifically, the methods provided herein are used to identify with confidence one or more lymphocyte receptor chain pairs in a sample, for example one or more functional chain pairs.