**4.2. Sex chromosome aneuploidies**

**3.2. Targeted sequencing**

**3.3. SNP-based sequencing**

villous sampling.

**4.1. Trisomy 21, 13, and 18**

ratio approach to calculate the risk for aneuploidy.

412 Next Generation Sequencing - Advances, Applications and Challenges

Targeted sequencing sequences only the regions / chromosome of interest and thus can be more time- and cost-efficient compared to whole genome sequencing. The principle is to selectively amplify the regions from chromosome 21, 13, and 18 followed by NGS. This method is also referred to as digital analysis of selected regions (DANSR). The amount of sequencing for a reliable detection is significantly lower around 40,000 and 1 million mapped sequences / sample. Unique to this type, the analysis uses a fetal fraction optimized risk score (FORTE) and takes into account the a priori risk (maternal age and gestational age) and uses an odds

This third approach was the most recent method introduced to the variety of clinically available NIPT options. This technique involves targeted amplification and sequencing of singlenucleotide polymorphisms (SNPs). SNPs are single base pairs that occur approximately once / 300 base pairs on the human genome and can be used to distinguish individuals. In addition to the above mentioned applications, maternal and fetal DNA also can be distinguished by SNP analysis. For this analysis, both maternal DNA from white cells from the buffy coat and maternal plasma which includes fetal and maternal DNA are used. In the SNP-technology originally introduced by Zimmermann et al. [16], 19,488 SNPs on the chromosomes 21, 13, 18, X, and Y are analyzed simultaneously. Taking into account the parental genotype, the fetal fraction, and the fetal chromosome copy number, billions of possible genotypes at a specific locus are considered by a complex algorithm and the observed allele distributions are com‐ pared to the expected allele distributions. By this method, the most likely fetal genotype can be calculated and a specific risk score for the analyzed aneuploidies is reported [16–19].

**4. Evidence on the quality of NIPT from published literature**

The initial studies on test quality for the most common aneuploidies were performed in highrisk collectives and focused on the sensitivities and specificities of the different cell-free DNA tests [20–26]. After the rapid clinical application of NIPT including many women at low risk, there was a demand for information on the positive predictive value of each individual test. The positive predictive value then was found to vary widely depending on the investigated cohort and could be as low as 45.4% for trisomy 21 [27], meaning that when a NIPT-test was positive only 45.4% of the fetuses were affected. This underlines the fact that although cell-free DNA testing performs better than the previous screening algorithms for aneuploidy, a positive test result requires confirmation with an invasive procedure such as amniocentesis or chorionic

The data for the three most common aneuploidies now stem from a number of large-scale studies from mainly high-risk collectives. The detection rate for trisomy 21 ranged from 97.5%

While reporting of fetal gender is feasible with cell-free DNA testing with high sensitivities of more than 95%, the reporting of sex chromosomal aneuploidies is more challenging. The most common sex chromosomal aneuploidies are 45X0 (Turner syndrome), XXX (Triple X syn‐ drome), XXY (Klinefelter syndrome), and XYY (Jacob syndrome).

While Turner syndrome can be detected on prenatal ultrasound, the others typically do not show sonographic signs but have been detected incidentally if an invasive procedure was performed for another reason. Compared to the most common aneuploidies, the detec‐ tion rates of sex chromosomal aneuploidies have lower specificities leading to higher false positive rates [23,33]. This is most likely due to the guanine–cytosine content of the X chromosome, which affects the reliability and accuracy of the sequencing data, the small size of the Y chromosome, and the sequence similarity between the X and the Y chromo‐ some. Furthermore, an unknown maternal or fetal mosaicism can interfere with the quantifications of the chromosomal representations. The reported numbers on detected sex chromosome aneuploidies other than Turner syndrome are very low with less than seven cases of each aneuploidy per study [23,34–36] so that reliable data are not present to date. The data on Turner syndrome need to be interpreted with caution since there may be a bias toward the non-viable cases and those detected with sonography. Furthermore, the follow-up data on test negative cases might be incomplete due to the fact that children with Turner syndrome might not show a noticeable phenotype at birth. Also, the rate of tests that do not receive a result due to difficulties with the interpretation of the sequencing data (non-reportables) seems to be higher compared to the autosomal aneuploidies. Taking into account some of these limitations, the detection rate for Turner syndrome ranges between 75% and 92% at a false positive rate of up to 0.3% [23,34–36].
