Development and analytical validation of a next-generation sequencing based microsatellite instability (MSI) assay
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Sarabjot Pabla1,*, Jonathan Andreas1,*, Felicia L. Lenzo1, Blake Burgher1, Jacob Hagen1, Vincent Giamo1, Mary K. Nesline1, Yirong Wang1, Mark Gardner1, Jeffrey M. Conroy1,2, Antonios Papanicolau-Sengos1, Carl Morrison1,2 and Sean T. Glenn1,2,3
1 OmniSeq Inc., Buffalo, NY 14203, USA
2 Center for Personalized Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA
3 Department of Molecular and Cellular Biology, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA
* These authors contributed equally to this work
|Sean T. Glenn,||email:||[email protected]|
Keywords: next-generation sequencing; NGS; microsatellite instability; MSI
Abbreviations: NGS: next-generation sequencing
Received: June 10, 2019 Accepted: July 29, 2019 Published: August 27, 2019
We have developed and analytically validated a next-generation sequencing (NGS) assay to classify microsatellite instability (MSI) in formalin-fixed paraffin-embedded (FFPE) tumor specimens.
The assay relies on DNA-seq evaluation of insertion/deletion (indel) variability at 29 highly informative genomic loci to estimate MSI status without the requirement for matched-normal tissue. The assay has a clinically relevant five-day turnaround time and can be conducted on as little as 20 ng genomic DNA with a batch size of up to forty samples in a single run.
The MSI detection method was developed on a training set (n = 94) consisting of 22 MSI-H, 24 MSS, and 47 matched normal samples and tested on an independent test set of 24 MSI-H and 24 MSS specimens. Assay performance with respect to accuracy, reproducibility, precision as well as control sample performance was estimated across a wide range of FFPE samples of multiple histologies to address pre-analytical variability (percent tumor nuclei), and analytical variability (batch size, run, day, operator). Analytical precision studies demonstrated that the assay is highly reproducible and accurate as compared to established gold standard PCR methodology and has been validated through NYS CLEP.
This assay provides clinicians with robust and reproducible NGS-based MSI testing without the need of matched normal tissue to inform clinical decision making for patients with solid tumors.
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