Evaluating Basic Next-Generation Sequencing Parameters in Relation to True/False Positivity Findings of Rare Variants in an Isolated Population from the Czech Republic South-Eastern Moravia Region with a High Incidence of Parkinsonism

Abstract

Next-generation sequencing in 16 genes known to be associated with parkinsonism, including coding DNA, intron/exon boundaries, and UTRs loci, was used to find rare variants in 30 patients and 12 healthy controls from an isolated population of South-Eastern Moravia in the Czech Republic where epidemiological data has proved a significantly increased prevalence of parkinsonism (2.9%). The aim of the study is to evaluate the true/false positivity ratio in relation to the basic sequencing parameters (coverage, type of mutation – SNV/INDEL, percentage of rare variants in heterozygosity, ± strand bias, and length of homopolymers). The final filtered rare variants were obtained from the Ion Torrent platform with the following workflow: Torrent Suite Base calling and BAM mapping, Ion Reporter Variant calling, and rare variant filtering. True positivity findings were distinguished from false by Sanger confirmation sequencing. In total, 36 rare variants (MAF ˂ 1%) were found, of which 50% were confirmed as true positive. For SNV, the probability of false positivity is 12%; for INDEL, the false positivity proportion is 84%. A high correlation in strand biases of reference and rare variants in heterozygous findings could be a very strong indicator for true positive variants.