Nested Primer Sets for High-Throughput Nucleic Acid Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for analyzing complex populations of nucleic acids, particularly with high-throughput sequencing platforms, face challenges such as uniform amplification and primer selection due to sequence variability caused by somatic hypermutation and clonal evolution, leading to limited sequence read lengths and declining sequence quality.
Innovation Solution
A method involving spatial isolation and sequencing of individual nucleic acid molecules, using nested sets of templates and bidirectional sequencing to generate clonotype profiles of T cell and B cell receptors, with amplification techniques like bridge PCR and sequencing-by-synthesis to maintain high-quality sequence reads despite sequence variability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high-throughput sequencing platforms are used to analyze complex nucleic acid populations, then sequencing throughput is improved, but sequence read length is limited and sequence quality declines
Solution Approach 1:
The patent segments the sequencing process by using multiple shorter sequence reads that overlap in their coverage of the target nucleic acid sequence. Instead of relying on a single long read, the method generates multiple reads from different positions, allowing the sequence to be reconstructed by combining overlapping regions. This segmentation enables high-throughput sequencing while maintaining sequence quality through redundant coverage.
Solution Approach 2:
The patent implements a nested amplification strategy where multiple sets of nested amplimers are generated from the original nucleic acid sequence. Each amplimer set produces sequence reads that overlap with adjacent reads, creating a nested structure of information. This nesting allows the sequence to be covered multiple times at different positions, improving both throughput and quality.
2Measurement precision
If uniform amplification of target populations is achieved, then sequence quality is improved, but primer selection becomes more difficult due to unknown target sequence variability
Solution Approach 1:
The patent employs parameter changes by using multiple primer sets with different binding specificities to target the same nucleic acid sequence. Each primer set is designed to bind to a different region or have different stringency, allowing the system to adapt to sequence variability. This multi-parameter approach ensures uniform amplification across diverse sequences without requiring prior knowledge of all possible variants.
Solution Approach 2:
The patent creates universal primer sets that can target multiple sequence variants simultaneously. By designing primers that bind to conserved regions or use degenerate bases, the same primer set can amplify sequences with unknown variability. This multi-functionality simplifies primer selection while maintaining sequence quality across diverse targets.
3Measurement precision
If sequence variability caused by somatic hypermutation and clonal evolution is accounted for, then clonotype profiling accuracy is improved, but amplification uniformity becomes more difficult to achieve
Solution Approach 1:
The patent applies dynamics by using multiple primer sets with varying binding characteristics to handle sequence variability dynamically. Instead of relying on a single static primer design, the system employs multiple primers that can adapt to different mutation states. This dynamic approach maintains amplification uniformity across sequences with somatic hypermutation and clonal evolution while preserving clonotype profiling accuracy.
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 approach enables the generation of accurate and reliable clonotype profiles with minimal amplification bias, allowing for the analysis of complex nucleic acid populations with improved sequence quality and length, even in regions with high variability.
Implementation Method 1
with amplification techniques like bridge PCR and sequencing-by-synthesis to maintain high-quality sequence reads
Implementation Method 2
amplification techniques like bridge PCR
Data Source
AI summary
The invention is directed to methods of generating sequence profiles of populations of nucleic acids, whose member nucleic acids contain regions of high variability, such as populations of nucleic acids encoding T cell receptors or B cell receptors. In one aspect, the invention provides pluralities of sets of primers for generating nested sets of templates from nucleic acids in such populations, thereby insuring the production of at least one template from which sequence reads are generated, despite such variability, or despite limited lengths or quality of sequence reads. In another aspect, members of such populations are bidirectionally sequenced so that further sequence information is obtained by analyzing overlapping sequence reads in the zones of highest variability.