title: A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene.
abstract: The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.
authors.affiliation: Diagnostic Genetics Unit, Careggi University Hospital, Florence, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Contini
authors.affiliation: Department of Biomedical Experimental and Clinical Sciences, Medical Genetics, University of Florence, Florence, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Paganini
authors.affiliation: Department of Biomedical Experimental and Clinical Sciences, Medical Genetics, University of Florence, Florence, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Sestini
authors.affiliation: Department of Biomedical Experimental and Clinical Sciences, Medical Genetics, University of Florence, Florence, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Candita
authors.affiliation: Department of Biomedical Experimental and Clinical Sciences, Medical Genetics, University of Florence, Florence, Italy.
authors.forename: Gabriele Lorenzo
authors.initials: GL
authors.lastname: Capone
authors.affiliation: Department of Biomedical Experimental and Clinical Sciences, Medical Genetics, University of Florence, Florence, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Barbetti
authors.affiliation: Diagnostic Genetics Unit, Careggi University Hospital, Florence, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Falconi
authors.affiliation: Diagnostic Genetics Unit, Careggi University Hospital, Florence, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Frusconi
authors.affiliation: Diagnostic Genetics Unit, Careggi University Hospital, Florence, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Giotti
authors.affiliation: Diagnostic Genetics Unit, Careggi University Hospital, Florence, Italy.
authors.forename: Costanza
authors.initials: C
authors.lastname: Giuliani
authors.affiliation: Diagnostic Genetics Unit, Careggi University Hospital, Florence, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Torricelli
authors.affiliation: Diagnostic Genetics Unit, Careggi University Hospital, Florence, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Benelli
authors.affiliation: Department of Biomedical Experimental and Clinical Sciences, Medical Genetics, University of Florence, Florence, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Papi
mesh_terms: D005787:Gene Frequency; D016515:Genes, Neurofibromatosis 2; D006801:Humans; D009030:Mosaicism; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D009154:Mutation; D016518:Neurofibromatosis 2 / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity