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Sequencing‐based microsatellite instability testing using as few as six markers for high‐throughput clinical diagnostics
Author(s) -
Gallon Richard,
Sheth Harsh,
Hayes Christine,
Redford Lisa,
Alhilal Ghanim,
O'Brien Ottilia,
Spiewak Helena,
Waltham Amanda,
McAnulty Ciaron,
Izuogu Osagie G.,
Arends Mark J.,
Oniscu Anca,
Alonso Angel M.,
Laguna Sira M.,
Borthwick Gillian M.,
SantibanezKoref Mauro,
Jackson Michael S.,
Burn John
Publication year - 2020
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.23906
Subject(s) - microsatellite instability , multiplex , microsatellite , biology , lynch syndrome , concordance , molecular inversion probe , computational biology , genetic marker , molecular marker , genetics , genotyping , colorectal cancer , cancer , dna mismatch repair , genotype , gene , allele
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.