OBJECTIVE The study aimed to validate a whole-genome sequencing-based NIPT laboratory method and our recently developed NIPTmer aneuploidy detection software with the potential to integrate the pipeline into prenatal clinical care in Estonia. METHOD In total, 424 maternal blood samples were included. Analysis pipeline involved cell-free DNA extraction, library preparation and massively parallel sequencing on Illumina platform. Aneuploidies were determined with NIPTmer software, which is based on counting pre-defined per-chromosome sets of unique k-mers from sequencing raw data. SeqFF was implemented to estimate cell-free fetal DNA (cffDNA) fraction. RESULTS NIPTmer identified correctly all samples of non-mosaic trisomy 21 (T21, 15/15), T18 (9/9), T13 (4/4) and monosomy X (4/4) cases, with the 100% sensitivity. However, one mosaic T18 remained undetected. Six false-positive (FP) results were observed (FP rate of 1.5%, 6/398), including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). The level of cffDNA of