Antigen-Specific BCR Sequencing via Barcode and UMI Correction
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Solution Overview
Problem
Current methods face challenges in accurately quantifying and identifying antigen-specific B-cell receptor (BCR) sequences due to amplification biases and difficulties in determining paired light and heavy chains from the same lymphoid cell, which hinders the correlation of BCR sequences with corresponding antigens or epitopes.
Innovation Solution
A method involving incubating B-cells with antigen libraries, isolating and sequencing nucleic acids, and using barcode and universal adapter sequences to enrich and identify antigen-specific BCR sequences through multiplex PCR and sequencing, allowing for the correlation of BCR sequences with specific antigens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplexed amplification is used to sequence diverse BCR gene templates, then sequencing throughput is improved, but amplification biases occur that skew quantitative data
Solution Approach 1:
The patent modifies the concentration and composition parameters of PCR primers to achieve uniform amplification across diverse BCR templates. By optimizing primer concentrations and using equimolar amounts of all primers in the multiplex reaction, the method reduces amplification biases while maintaining high sequencing throughput.
Solution Approach 2:
The patent introduces unique molecular identifiers (UMIs) as intermediary sequences that are incorporated during reverse transcription. These UMIs serve as traceable markers that allow computational correction of amplification biases, enabling accurate quantification even when multiplexed amplification is used.
2Productivity
If existing sequencing methodologies are used, then BCR repertoire profiling is possible, but it is difficult to determine that both chains originated from the same lymphoid cell
Solution Approach 1:
The patent merges the sequencing of heavy and light chains into a single multiplexed reaction by incorporating template-switching oligonucleotides that add universal adapter sequences to both chains. This allows both chains from the same BCR to be amplified and sequenced together, preserving their pairing information.
Solution Approach 2:
The patent uses template-switching oligonucleotides to create copies of the BCR gene templates with added adapter sequences. These copied sequences include unique molecular identifiers that are inherited by both heavy and light chain products, enabling traceability of their common origin through computational analysis.
3Adaptability or versatility
If diverse primers are used in multiplexed amplification, then coverage of BCR diversity is improved, but unequal use of primers creates artifacts that skew data
Solution Approach 1:
The patent systematically optimizes primer parameters including concentration, length, and sequence composition to minimize differential amplification. All primers are used at equimolar concentrations, and the method includes control experiments to identify and correct for any remaining primer biases in the quantitative analysis.
Solution Approach 2:
The patent incorporates computational feedback mechanisms that use the unique molecular identifiers to measure actual amplification efficiency for each primer. This feedback information is used to correct quantitative measurements, allowing the method to maintain high reliability even when using diverse primers for comprehensive BCR coverage.
Data Source
AI summary
Compositions and methods are disclosed for identifying B-cell receptor sequences that bind to corresponding antigens. The disclosed methods and related embodiments permit the identification paired relationships between rearranged gene segments of B-cell receptors with unique antigens.