Spike-In Oligo Libraries for Validating Patient-Specific Sequencing
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Solution Overview
Problem
Current methods for validating patient-specific oligonucleotides (oligos) in genomic sequencing are costly and time-consuming, limiting the practical application of personalized medicine, particularly in liquid biopsies for cancer monitoring.
Innovation Solution
A method involving the use of spike-in control oligos, where sets of spike-in sequences are introduced into a patient sample, cosynthesized with synthesis-control oligos, and assayed to determine the dose-response characteristics, providing a quality control check for patient-specific oligos.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If patient-specific oligos are used for targeted sequencing to reduce cost, then sequencing cost is reduced, but validation complexity and time increase significantly
Solution Approach 1:
The patent introduces synthesis control oligos as intermediary elements that are cosynthesized with patient-specific oligos but have known, predictable sequences. These control oligos serve as mediators to validate the synthesis process without requiring full validation of each patient-specific oligo, thereby reducing validation complexity while maintaining cost benefits
Solution Approach 2:
The patent performs preliminary validation by incorporating synthesis control oligos during the oligo synthesis process itself. By validating the synthesis process upfront using these control elements, the need for extensive post-synthesis validation of each patient-specific oligo is eliminated, reducing both time and complexity
2Measurement precision
If comprehensive testing is performed to identify all patient-specific mutations, then detection accuracy is improved, but testing cost increases significantly
Solution Approach 1:
The patent segments the testing process into two phases: initial comprehensive testing to identify patient-specific mutations, followed by targeted testing using patient-specific oligos for subsequent samples. This segmentation allows high accuracy in the initial phase while reducing costs in ongoing monitoring phases
Solution Approach 2:
The patent changes the testing parameters from comprehensive genome-wide sequencing to targeted sequencing using patient-specific oligos. This parameter change maintains detection accuracy for known mutations while significantly reducing testing costs for longitudinal monitoring
3Measurement precision
If multiple ctDNA targets are evaluated to increase sensitivity, then detection sensitivity is improved, but sequencing cost becomes prohibitive
Solution Approach 1:
The patent creates universal patient-specific oligos that can target multiple ctDNA markers simultaneously. These oligos are designed to capture multiple mutation sites in a single assay, providing multi-functionality that increases detection sensitivity while avoiding the need for separate sequencing experiments for each target
Data Source
AI summary
Compositions, kits, and methods are provided for validating patient-specific oligonuclotide assays using spike-in controls. The compositions comprise multiple sets of spike-in oligos, each set including a unique reference sequence and near-neighbor sequences with at least one substitution, insertion, or deletion. Near-neighbor concentrations are less than the reference and collectively span at least four orders of magnitude (e.g., 1:10 to 1:10,000); all sets have the same number of near-neighbors and concentration ratios. Spike-in sequences are unrelated to human sequences and are complementary to synthesis-control oligos at known concentrations. Kits further include synthesis-control oligos and patient-specific oligos for human DNA or RNA targets. Methods involve spiking a sample, co-synthesizing control and patient-specific oligos, assaying by PCR or sequencing, and validating limit of detection and dynamic range by comparing observed counts to known ratios. Applications include ctDNA and cfRNA testing.