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Sequencing‐based microsatellite instability testing using as few as six markers for high‐throughput clinical diagnostics

Lookup NU author(s): Dr Richard Gallon, Dr Harsh Sheth, Christine Hayes, Lisa Redford, Dr Ghanim Alhilal, Ottilia O'Brien, Dr Helena Spiewak, Dr Ciaron McAnulty, Dr Osagie Izuogu, Dr Gillian Borthwick, Dr Mauro Santibanez Koref, Dr Michael Jackson, Professor Sir John BurnORCiD



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer‐predisposition, and can be used to predict response to immunotherapy. Here, we present a single‐molecule molecular inversion probe and sequencing‐based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward–forward stepwise selection was used to identify a 6‐marker subset of equal accuracy to the 24‐marker panel. Assessment of assay detection limits showed that the 24‐marker panel is marginally more robust to sample variables than the 6‐marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.

Publication metadata

Author(s): Gallon R, Sheth H, Hayes C, Redford L, Alhilal G, O'Brien O, Spiewak H, Waltham A, McAnulty C, Izuogu OG, Arends MJ, Oniscu A, Alonso AM, Laguna SM, Borthwick GM, Santibanez-Koref M, Jackson MS, Burn J

Publication type: Article

Publication status: Published

Journal: Human Mutation

Year: 2020

Volume: 41

Issue: 1

Pages: 332-341

Print publication date: 24/12/2019

Online publication date: 31/08/2019

Acceptance date: 26/08/2019

Date deposited: 09/12/2019

ISSN (print): 1059-7794

ISSN (electronic): 1098-1004

Publisher: Wiley


DOI: 10.1002/humu.23906

PubMed id: 31471937


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Funder referenceFunder name
Cancer Research UK