**3. Technical principles of the clinically available Non-Invasive Prenatal Tests (NIPT)**

In the following passage, we will focus on the basic principles of the commercially available cell-free DNA test that offers analysis for the three most common aneuploidies today. Basically, there are three different types of approaches of prenatal cell-free DNA testing: whole genome sequencing, targeted genome sequencing, and single-nucleotide polymorphism (SNP)-based sequencing. Another fourth approach, epigenetic testing of fetal DNA methylation, which is not yet clinically available, has shown promising results. It detects fetal-specific epigenetic patterns and unique methylation profiles [14,15].

All techniques use massive parallel genomic sequencing (MPS) or NGS, which refers to the high-throughput DNA sequencing technology that can sequence millions of DNA molecules in parallel [13]. For prenatal testing, both cell-free DNA of maternal and fetal origin present in maternal peripheral blood are sequenced and these fragments are mapped to a reference chromosome. It is important to keep in mind that the majority of sequenced DNA is of maternal origin and that the difference between a normal fetus and fetus with an additional chromosome will only show a slight increase compared to a normal reference chromosome since the aneuploid part forms only about 10% of the sequenced DNA. Quantitative accuracy of the applied method, therefore, is crucial to exclude an aneuploidy. A minimum percentage of fetal DNA is required to reliably perform an analysis and is usually set at a minimum of 4%.
