Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Gallon R

PROVIDER: S-EPMC6973255 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Gallon Richard R Sheth Harsh H Hayes Christine C Redford Lisa L Alhilal Ghanim G O'Brien Ottilia O Spiewak Helena H Waltham Amanda A McAnulty Ciaron C Izuogu Osagie G OG Arends Mark J MJ Oniscu Anca A Alonso Angel M AM Laguna Sira M SM Borthwick Gillian M GM Santibanez-Koref Mauro M Jackson Michael S MS Burn John J

Human mutation 20190915 1

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 ...[more]

PMID: 31471937

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Project description:BackgroundBovine tuberculosis (bTB) is an enduring contagious disease of cattle that has caused substantial losses to the global livestock industry. Despite large-scale eradication efforts, bTB continues to persist. Current bTB tests rely on the measurement of immune responses in vivo (skin tests), and in vitro (bovine interferon-γ release assay). Recent developments are characterized by interrogating the expression of an increasing number of genes that participate in the immune response. Currently used assays have the disadvantages of limited sensitivity and specificity, which may lead to incomplete eradication of bTB. Moreover, bTB that reemerges from wild disease reservoirs requires early and reliable diagnostics to prevent further spread. In this work, we use high-throughput sequencing of the peripheral blood mononuclear cells (PBMCs) transcriptome to identify an extensive panel of genes that participate in the immune response. We also investigate the possibility of developing a reliable bTB classification framework based on RNA-Seq reads.Methodology/principal findingsPooled PBMC mRNA samples from unaffected calves as well as from those with disease progression of 1 and 2 months were sequenced using the Illumina Genome Analyzer II. More than 90 million reads were splice-aligned against the reference genome, and deposited to the database for further expression analysis and visualization. Using this database, we identified 2,312 genes that were differentially expressed in response to bTB infection (p<10(-8)). We achieved a bTB infected status classification accuracy of more than 99% with split-sample validation on newly designed and learned mixtures of expression profiles.Conclusions/significanceWe demonstrated that bTB can be accurately diagnosed at the early stages of disease progression based on RNA-Seq high-throughput sequencing. The inclusion of multiple genes in the diagnostic panel, combined with the superior sensitivity and broader dynamic range of RNA-Seq, has the potential to improve the accuracy of bTB diagnostics. The computational pipeline used for the project is available from http://code.google.com/p/bovine-tb-prediction.

Dataset Information

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

Publications

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

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