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MSIanalyzer: Targeted Nanopore Sequencing Enables Single Nucleotide Resolution Analysis of Microsatellite Instability
Ting Zhai1, Daniel J Laverty1, Zachary D Nagel1
1Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA 02115.
Biorxiv : the Preprint Server for Biology
|July 16, 2025
Summary
We developed a new sequencing method to precisely measure microsatellite instability (MSI) in cancer. This technique profiles MSI at single-nucleotide resolution, offering improved accuracy for research and potential clinical applications.
Area of Science:
- Genomics
- Molecular Biology
- Cancer Research
Background:
- Microsatellite instability (MSI) is a key biomarker in cancer, particularly colorectal cancer.
- Accurate profiling of MSI is crucial for understanding cancer mechanisms and developing diagnostics.
- Existing methods for MSI analysis have limitations in resolution and capturing allelic diversity.
Purpose of the Study:
- To develop and validate a targeted sequencing pipeline for high-resolution microsatellite instability profiling.
- To assess the performance of the pipeline across various cancer cell lines and healthy donor samples.
- To establish a quantitative method for MSI analysis that preserves read-level diversity.
Main Methods:
- Targeted sequencing of five Bethesda panel microsatellite loci using Oxford Nanopore Technology.
- Development of an anchor-extension algorithm for capturing repeat motifs with interruptions.
- Application of cluster-aware Dirichlet-multinomial and beta-binomial tests for statistical analysis.
Main Results:
- The pipeline successfully profiled MSI at single-nucleotide resolution in colorectal cancer cell lines and healthy donor samples.
- Distinct repeat profiles were identified in microsatellite unstable cell lines (HCT15, HCT116) compared to stable cell lines (TK6, U2OS) and healthy controls.
- Allelic diversity at different MSI loci was uncovered across the analyzed samples.
Conclusions:
- The developed targeted sequencing pipeline provides a robust method for quantitative MSI profiling.
- This approach complements existing short tandem repeat callers by preserving read-level diversity.
- The method holds potential for advancing mechanistic cancer research and clinical assay development for MSI detection.
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