**3.1. Whole genome sequencing**

technique of next-generation sequencing (NGS) that lead to the transfer of this research into clinical practice. After the clinical availability and introduction of cell-free DNA analysis for the most common fetal aneuploidies (Trisomy 21, 13, and 18) in 2011, there has been an extremely high demand by pregnant women and to date approximately 1.4 million analyses have been performed worldwide assuming that there will be around 1 million/year in 2015 [2]. Most current tests count DNA fragments, map them to the chromosomes, and quantitatively compare the cell-free-DNA in maternal blood with a euploid reference genome. This new screening tool in prenatal diagnostics has marked the beginning of a new era in prenatal care and has significantly reduced the rate of invasive prenatal procedures such as chorionic villous

With the broad availability of non-invasive prenatal genetic testing, a number of new issues have emerged concerning its reasonable clinical application, ethical concerns, integration into current public healthcare plans, counseling issues, and the role of prenatal ultrasound screening. In the following, we will discuss the current and future concepts of prenatal cellfree fetal DNA testing and show the current impact on clinical care among different risk groups

The idea that genetic information of the fetus can be discovered by investigating maternal blood during pregnancy stems from the historic concept of Georg Schmorl, who described cross-placental trafficking of fetal cells into the maternal circulation. Fetal trophoblast cells were first demonstrated in lung tissue in mothers who died from eclampsia [3]. The isolation of fetal cells has remained a challenge due to their very low quantity [4,5], the limited knowl‐ edge on the characteristics and suboptimal markers for identification [6]. The focus has moved to the analysis of fetal cell-free DNA fragments which were first described in 1997 [1]. Cellfree DNA in maternal blood is comprised of extracellular DNA fragments that can be found in the maternal plasma and serum. The majority of cell-free DNA in maternal circulation is of maternal origin and around 10% is of fetal origin. Cell-free fetal DNA is released into the maternal circulation from cells of the placenta. It can be detected very early in pregnancy and

Initially, it was only feasible to analyze sequences of paternal origin and de novo mutations that were different from the maternal genome due to the high percentage of maternal cell-free DNA. Therefore, early studies focused on fetal Rhesus-status and on the detection of autoso‐ mal-dominant disorders of paternal inheritance [8]. Real-time quantitative PCR technology proved to be suitable for the detection of fetal loci that are different from the maternal genome such as the Y chromosome. Fetal gender determination was applied in families with a high risk for X-chromosome-linked disorders in which only male fetuses are affected from the disease and for the detection of fetal Rhesus D in pregnancies at risk for hemolytic disease of the newborn [9–11]. Just recently, non-invasive prenatal testing for routine fetal Rhesus D genotyping in Rhesus-negative women has been proven to be highly accurate over a 2-year

sampling and amniocentesis.

taking into account medical, social, and ethical aspects.

410 Next Generation Sequencing - Advances, Applications and Challenges

**2. Fetal cells and cell-free DNA**

is cleared a few hours after birth [7].

For this analysis, the entire cell-free DNA is sequenced in short reads and compared to a reference human genomic database and each sequence is matched to a specific chromosome. The counts observed in the individual probe are then compared to an euploid reference sample. If the fetus carries an additional chromosome (as in trisomy 21, 13, and 18), more fragments are expected for the additional chromosome compared with a normal fetus. However, it is necessary to sequence many millions of DNA fragments (12–15 × 106 mapped sequences) to ensure that there are sufficient chromosome fragments (reads) from the specific chromosome to detect statistically significant differences between aneuploid and euploid fetuses. Also, there are several other aspects of sequencing and the fetal fraction as well as the guanine–cytosine content, etc. that need to be taken into account.
