NGS Probe Concentration Tuning for Uniform Sequencing Coverage
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Solution Overview
Problem
Existing methods for designing probes for next-generation sequencing assays are inadequate in optimizing probe concentrations for specific samples, leading to uneven coverage and inefficiencies in sequencing processes.
Innovation Solution
The method involves adjusting the concentration of over-performing probes by altering the ratio of labeled and unlabeled probes, using techniques such as adding locked nucleic acid modifications, hairpins, and interfering oligos to achieve optimized probe sets with even capture rates, combined with methods to reduce amplification of certain RNA or DNA molecules during sequencing library generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If uniform probe concentration is used across all probes, then probe pool simplicity is maintained, but sequencing coverage uniformity deteriorates due to over-performing probes capturing excessive targets
Solution Approach 1:
The patent applies local quality by assigning different concentrations to different probes based on their individual performance characteristics. Over-performing probes are assigned lower concentrations while under-performing probes receive higher concentrations, creating a non-uniform concentration distribution that optimizes overall sequencing coverage uniformity.
Solution Approach 2:
The patent changes the concentration parameter of probes based on their performance metrics. By calculating performance scores and adjusting concentrations accordingly, the system transforms a uniform probe pool into an optimized pool where each probe's concentration is tailored to its specific performance characteristics.
2Manufacturing precision
If reverse complement probes are added to reduce over-performing probe concentration, then coverage uniformity improves, but probe pool complexity increases
Solution Approach 1:
The patent uses copying by creating reverse complement versions of over-performing probes. These reverse complement copies are added to the probe pool at concentrations designed to cancel out the excessive capture activity of the original over-performing probes, thereby reducing coverage uniformity issues while managing complexity through a systematic copying approach.
3Measurement precision
If probe concentrations are optimized for specific samples, then sequencing accuracy improves, but assay adaptability decreases
Solution Approach 1:
The patent applies preliminary action by pre-calculating performance scores for each probe based on reference genome data and expected sample characteristics. This allows the system to pre-determine optimal concentrations before actual sequencing, enabling sample-specific optimization while maintaining a systematic approach that can be adapted to different sample types.
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 results in highly optimized probe sets that provide improved sequencing coverage uniformity and depth across the genome, enhancing the accuracy and efficiency of nucleic acid capture and sequencing.
Implementation Method 1
The method involves adjusting the concentration of over-performing probes by altering the ratio of labeled and unlabeled probes, using techniques such as adding locked nucleic acid modifications, hairpins, and interfering oligos to achieve optimized probe sets with even capture rates
Data Source
AI summary
Systems and methods are provided for determining an optimized probe set. The method proceeds by obtaining a set of probes, where each probe has a respective concentration. The set of probes is assayed against a sample library, and at least i) a respective recovery rate for each probe in the set of probes, and ii) a median recovery rate for the set of probes are obtained. Modify the respective concentration of each probe that does not satisfy predetermined recovery rate threshold. Reevaluate the set of probes against the sample library. Repeat the modifying and reevaluation until the respective updated recovery rate for each probe in the updated set of probes satisfies the predetermined recovery rate threshold, thereby providing the optimized set of probes for the sample library.