Clonality Detection via Parallel Sequencing of Immune Receptor Genes
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Solution Overview
Problem
Current methods for detecting clonality in immune receptor genes are limited in sensitivity and specificity, often resulting in indeterminate results and requiring labor-intensive processes, which hinder the accurate analysis of clonal populations in healthy and malignant immune responses.
Innovation Solution
The method involves parallel sequencing of DNA samples from complex cell populations, using bar-coded primers to amplify and sequence hypervariable regions of immune receptor genes, allowing for the detection of coincident sequences across replicates, which indicates clonal expansion, and providing a highly sensitive and consistent assay for measuring clonal populations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional methods (Southern blotting, PCR sizing, light chain analysis) are used to detect clonality, then the assay can be performed with standard equipment, but the measurement precision and reliability are insufficient, yielding indeterminate results
Solution Approach 1:
The patent replaces traditional mechanical/chemical detection methods (Southern blotting, PCR sizing) with high-throughput sequencing technology. This substitution enables direct reading of immune receptor gene sequences at base-pair resolution, providing precise identification of clonal expansions that were indeterminate with previous methods.
Solution Approach 2:
The patent changes the detection parameter from indirect signal measurement (band intensity, fragment size) to direct sequence analysis. By sequencing the hypervariable regions of immune receptor genes, the method achieves precise measurement of clonal populations through sequence frequency analysis, transforming the measurement paradigm entirely.
2Measurement precision
If deep sequencing of immune receptor genes is performed to detect clonal expansions, then measurement precision improves, but the device complexity and cost increase
Solution Approach 1:
The patent segments the immune receptor gene into specific hypervariable regions (CDR1, CDR2, CDR3) for targeted sequencing. By focusing only on these critical regions rather than sequencing entire genes, the method reduces sequencing depth requirements and computational complexity while maintaining high measurement precision for clonal detection.
Solution Approach 2:
The patent develops a universal sequencing approach that can detect various types of clonal expansions (malignant clones, vaccinated clones, autoimmune clones) using the same methodology. This multi-functional assay reduces overall system complexity by eliminating the need for multiple specialized tests.
3Productivity
If traditional clonality assays are used, then the analysis can be completed with standard laboratory equipment, but the productivity is low due to labor-intensive processes
Solution Approach 1:
The patent replaces manual, labor-intensive laboratory procedures with automated high-throughput sequencing. The process transitions from hands-on Southern blotting and manual PCR analysis to automated sequence reading and computational analysis, dramatically increasing productivity while reducing labor requirements.
Solution Approach 2:
The patent uses digital sequencing data as copies of the biological sample information. Instead of physically manipulating and analyzing multiple samples through tedious manual processes, the method creates digital sequences that can be rapidly copied, stored, and analyzed computationally, enabling high-throughput processing.
4Measurement precision
If sensitive clonal detection is performed to detect small clonal populations, then measurement precision improves, but the difficulty of detecting and measuring increases
Solution Approach 1:
The patent changes the detection parameter to sequence frequency analysis. By counting the frequency of specific sequences in the high-throughput sequencing data, the method can detect small clonal populations through statistical analysis of sequence distributions, making even rare clones measurable through straightforward computational methods rather than difficult experimental techniques.
Data Source
AI summary
Methods are provided for the detection and analysis of clonality in a cell population, where parallel sequencing is applied to a nucleic acid sample obtained from the cell population, optionally a population of lymphocytes. Replicate samples are amplified, and sequenced, where identification of coincident sequences in two or more replicates is indicative of clonal expansion.