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Background Introduction of cell‐free fetal DNA (cff‐DNA) testing in maternal blood opened possibilities to improve the performance of combined first‐trimester screening (cFTS) in terms of better detection of trisomies and lowering invasive testing rate. The use of new molecular methods, such as chromosomal microarray analysis (CMA) and next‐generation sequencing (NGS), has shown benefits in prenatal diagnosis of chromosomal and genetic diseases, which are not detectable with cff‐DNA screening, but require an invasive procedure. Methods The objective of this study was to evaluate prospectively during two years performance of CMA and NGS in high‐risk pregnancies. Initially, we investigated 14,566 singleton pregnancies with cFTS. A total of 334 high‐risk pregnancies were selected for CMA diagnostic performance evaluation and 28 cases of highly dysmorphic fetuses for NGS analysis. CMA study group was divided into two groups based on the indications for testing; group A patients with high‐risk for trisomies after cFTS, but normal ultrasound and group B patients who met criteria for CMA as a first‐tier diagnostic test. Results The diagnostic yield of CMA was overall 3.6% (1.6% in Group A and 6.0% in Group B). In NGS analysis group, we report diagnostic yield of 17.9%. Conclusion The use of CMA in high‐risk pregnancies is justified and provides relevant clinical information in 3.6% of the cases. NGS analysis in fetuses with multiple anomalies shows promising results, but more investigations are needed for a better understanding of practical applications of this molecular diagnosis method in prenatal settings.

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

Non-invasive prenatal testing (NIPT) is a recent and rapidly evolving method for detecting genetic lesions, such as aneuploidies, of a fetus. However, there is a need for faster and cheaper laboratory and analysis methods to make NIPT more widely accessible. We have developed a novel software package for detection of fetal aneuploidies from next-generation low-coverage whole genome sequencing data. Our tool – NIPTmer – is based on counting pre-defined per-chromosome sets of unique k-mers from raw sequencing data, and applying linear regression model on the counts. Additionally, the filtering process used for k-mer list creation allows one to take into account the genetic variance in a specific sample, thus reducing the source of uncertainty. The processing time of one sample is less than 10 CPU-minutes on a high-end workstation. NIPTmer was validated on a cohort of 583 NIPT samples and it correctly predicted 37 non-mosaic fetal aneuploidies. NIPTmer has the potential to reduce significantly the time and complexity of NIPT post-sequencing analysis compared to mapping-based methods. For non-commercial users the software package is freely available at http://bioinfo.ut.ee/NIPTMer/.

ObjectiveThe study aimed to validate a whole-genome sequencing-based NIPT method and our newly developed NIPTmer analysis software with the potential to integrate the pipeline into prenatal clinical care in Estonia.MethodIn total, 447 maternal blood samples were included to the study. Analysis pipeline involved whole-genome library preparation and massively parallel sequencing on Illumina NextSeq 500. Aneuploidy status was determined with NIPTmer software, which is based on counting pre-defined per-chromosome sets of unique k-mers from raw sequencing data. To estimate fetal fraction (FF) from total cell-free DNA SeqFF was implemented.ResultsNIPTmer software allowed to identify correctly all samples of non-mosaic T21 (15/15), T18 (9/9) and T13 (4/4) cases. However, one mosaic T18 remained undetected. Six false positive results were observed, including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). FF < 4% (2.8-3.99%) was estimated in eight samples, including two samples with T13 and T18. Despite low FF, these two samples were determined as aneuploid with NIPTmer software.ConclusionOur NIPT analysis pipeline proved to perform efficiently in detecting common fetal aneuploidies T21, T18 and T13 and is feasible for implementation into clinical service in Estonia.