Targeted Sequencing Structural Variation Mapping
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Solution Overview
Problem
Next-generation sequencing (NGS) platforms face challenges in accurately mapping sequences with large structural variations, such as inversions and translocations, and distinguishing clinically relevant genes from pseudogenes due to limitations in read lengths and haplotype reconstructions.
Innovation Solution
A method involving the use of primers and blocking elements to sequence multiple regions of a nucleic acid template, combined with interposing oligonucleotide probes and terminal probes to form an integrated strand complement, enabling accurate sequencing of long sequences and resolving structural variations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current NGS platforms with read lengths of 35-150 base pairs are used, then high-throughput massively parallel sequencing can be achieved, but accurate mapping of sequences with large structural variations cannot be accomplished
Solution Approach 1:
The patent divides the sequencing process into multiple independent sequencing reactions, each targeting a specific region of the template nucleic acid. By using multiple primers that anneal to different regions and performing separate extension reactions for each, the system can reconstruct long sequences with structural variations by combining information from multiple segmented reads, thereby achieving both high throughput and accurate mapping of structural variations.
2Productivity
If current NGS platforms with read lengths of 35-150 base pairs are used, then high-throughput massively parallel sequencing can be achieved, but accurate haplotype reconstructions cannot be performed
Solution Approach 1:
The patent segments the template nucleic acid into multiple regions that can be independently sequenced while maintaining information about their relative positions and phased relationships. By performing multiple sequencing reactions with different primers targeting specific regions and combining the results, the system preserves haplotype information that would be lost in conventional single-read approaches, enabling accurate haplotype reconstructions alongside high-throughput sequencing.
3Measurement precision
If multiple sequencing reactions are performed on different regions of the template nucleic acid, then accurate mapping of long sequences can be achieved, but sequencing time and process complexity increase
Solution Approach 1:
The patent performs preliminary actions by designing and annealing multiple region-specific primers to the template nucleic acid before the actual sequencing reactions. This pre-positioning of primers at specific regions allows subsequent sequencing reactions to proceed efficiently and independently, reducing the overall time required compared to sequential approaches, while maintaining high mapping accuracy for long sequences through the combined data from multiple primed regions.
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 enhances the ability to map long sequences with structural variations and differentiate between clinically relevant genes and pseudogenes, improving haplotype reconstructions and sequencing accuracy.
Implementation Method 1
incorporating with a polymerase one or more nucleotides into the first primer to create a first extension strand
Implementation Method 2
contacting the template nucleic acid with a blocking element thereby terminating extension of the first extension strand
Implementation Method 3
hybridizing one or more interposing oligonucleotide probes to the template nucleic acid
Data Source
AI summary
Disclosed herein, inter alia, are methods of making, amplifying, and sequencing compositions, and kits useful in obtaining sequencing data.