OS-Seq Tumor Microsatellite Profiling for MSI and EMAST Detection
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Solution Overview
Problem
Current methods for detecting microsatellite instability (MSI) in cancer, particularly colorectal carcinoma (CRC), are limited as they primarily focus on mono- and dinucleotide repeats, overlooking instability in tetranucleotide repeats and missing important genetic features.
Innovation Solution
A new sequencing approach that profiles instability across different classes of microsatellites and cancer genes, including mono-, di-, tri-, and tetranucleotide repeats, with ultra-depth sequencing and minimal amplification error to accurately detect somatic microsatellite and gene mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR-based MSI testing is used, then mono- and dinucleotide microsatellite instability can be detected, but tetranucleotide microsatellite instability (EMAST) is missed
Solution Approach 1:
The patent applies universality by designing a PCR assay that can detect multiple classes of microsatellite repeats (mono-, di-, tri-, and tetranucleotide) within a single testing framework. The method uses universal primers that can bind to various microsatellite classes, enabling the system to perform multiple detection functions simultaneously rather than requiring separate assays for each microsatellite class.
Solution Approach 2:
The patent segments the microsatellite detection process by creating separate primer sets and analysis protocols for different microsatellite classes. Each microsatellite class (mono-, di-, tri-, tetranucleotide) is analyzed independently through specific primer combinations, allowing precise detection of EMAST while maintaining the ability to detect traditional MSI markers. This segmentation enables comprehensive coverage without compromising the accuracy for any specific class.
2Measurement precision
If PCR amplification is performed to detect MSI, then microsatellite allele size changes can be identified, but PCR stutter artifacts complicate the detection particularly when allelic shift is less than 3 bp
Solution Approach 1:
The patent applies local quality by optimizing PCR conditions specifically for microsatellite amplification to minimize stutter artifacts. This includes using specialized polymerases with proofreading activity, adjusting annealing temperatures, and employing hot-start PCR techniques. These localized optimizations reduce the harmful stutter effect without compromising the ability to detect small allelic shifts of less than 3 bp.
Solution Approach 2:
The patent replaces traditional capillary electrophoresis-based size analysis with next-generation sequencing technology. This substitution eliminates the mechanical limitations of electrophoresis resolution and allows for direct sequencing of microsatellite regions, providing base-pair level precision in detecting allelic shifts while avoiding the stutter artifact problem that plagues PCR-based size detection methods.
3Productivity
If targeted sequencing with gene panels is used, then MSI can be detected through exon-based mono- or dinucleotide repeats, but tetranucleotide microsatellites are not included in the sequencing targets
Solution Approach 1:
The patent segments the targeted sequencing approach by designing specific bait sets or capture probes that target different microsatellite classes separately. This allows the sequencing platform to efficiently process and analyze mono-, di-, tri-, and tetranucleotide repeats in distinct analytical pipelines, maintaining high throughput while achieving comprehensive microsatellite class coverage including EMAST regions.
Solution Approach 2:
The patent creates a universal targeted sequencing panel that incorporates binding targets for all four microsatellite classes. The same sequencing library preparation and initial sequencing workflow handles all microsatellite types, with downstream bioinformatics analysis branching into class-specific evaluation. This multi-functional design maintains productivity while expanding versatility to include tetranucleotide microsatellites.
Data Source
AI summary
A method for determining if a tumor has a mutation in a microsatellite is provided. In some embodiments, the method may comprise: (a) isolating genomic DNA from a tumor sample and a non-tumor sample from the same patient to produce: i. a sample of tumor DNA and ii. a sample of non-tumor DNA, respectively, (b) without pre-amplifying the tumor or non-tumor DNA, sequencing a plurality of microsatellite loci from both the tumor and non-tumor DNA using OS-seq to provide sequence reads, wherein the sequenced microsatellite loci comprise mononucleotide, dinucleotide, trinucleotide and tetranucleotide microsatellites loci, and (c) comparing the results.