Noninvasive prenatal testing by massive parallel sequencing of maternal plasma DNA has rapidly been adopted as a mainstream method for detection of fetal trisomy 21, 18 and 13. Despite the relative high accuracy of current NIPT testing, a substantial number of false-positive and false-negative test results remain. Here, we present an analysis pipeline, which addresses some of the technical as well as the biologically derived causes of error. Most importantly, it differentiates high z-scores due to fetal trisomies from those due to local maternal CNVs causing false positives. This pipeline was retrospectively validated for trisomy 18 and 21 detection on 296 samples demonstrating a sensitivity and specificity of 100%, and applied prospectively to 1350 pregnant women in the clinical diagnostic setting with a result reported in 99.9% of cases. In addition, values indicative for trisomy were observed two times for chromosome 7 and once each for chromosomes 15 and 16, and once for a segmental trisomy 18. Two of the trisomies were confirmed to be mosaic, one of which contained a uniparental disomy cell line. As placental trisomies pose a risk for low-grade fetal mosaicism as well as uniparental disomy, genome-wide noninvasive aneuploidy detection is improving prenatal management. INTRODUCTIONThe presence of circulating cell-free fetal DNA in the maternal plasma of the pregnant woman, 1 in combination with recent advances in massively parallel sequencing (MPS) technologies, has made noninvasive prenatal testing (NIPT) of fetal aneuploidy a reality. NIPT reduces the need for invasive sampling and the associated risk of procedure-related pregnancy loss. In 2008, it was demonstrated that noninvasive fetal aneuploidy detection by MPS was feasible. 2,3 Multiple clinical validation studies using either targeted or whole-genome sequencing demonstrated the high sensitivity and specificity of NIPT. [4][5][6][7][8][9][10][11][12][13][14][15] Although most validation studies were predominantly evaluating the clinical validity in pregnancies at increased risk of the most common aneuploidies, it was recently shown that screening all pregnant women has positive predictive values of 45.5% and 40% for detection of trisomies 21 and 18, respectively. 16 MPS for aneuploidy detection applies counting statistics to millions of sequencing reads to identify subtle changes in the small percentage of fetal DNA present in the total cell-free DNA isolated from maternal plasma. 17,18 An increase or decrease in the number of normalized sequencing reads, typically converted to a 'z-score', 18 a 'normalized chromosome value', 13 genome-wide normalized score 19 or by 'withinsample copy number aberration detector' 20 is indicative of aneuploidy for the respective chromosome. Despite the high accuracy of current NIPT testing, a baseline false-positive and false-negative rate remains. Those incorrect results may have both biological and technical causes:

This approach permits the non-invasive detection of fetal autosomal aneuploidies and identifies pregnancies with a high risk of fetoplacental mosaicism. Knowledge about the presence of chromosomal mosaicism in the placenta influences risk estimation, genetic counseling, and improves prenatal management.

We analyzed by next-generation sequencing (NGS) 67 epilepsy genes in 19 patients with different types of either isolated or syndromic epileptic disorders and in 15 controls to investigate whether a quick and cheap molecular diagnosis could be provided. The average number of nonsynonymous and splice site mutations per subject was similar in the two cohorts indicating that, even with relatively small targeted platforms, finding the disease gene is not an univocal process. Our diagnostic yield was 47% with nine cases in which we identified a very likely causative mutation. In most of them no interpretation would have been possible in absence of detailed phenotype and familial information. Seven out of 19 patients had a phenotype suggesting the involvement of a specific gene. Disease-causing mutations were found in six of these cases. Among the remaining patients, we could find a probably causative mutation only in three. None of the genes affected in the latter cases had been suspected a priori. Our protocol requires 8-10 weeks including the investigation of the parents with a cost per patient comparable to sequencing of 1-2 medium-to-large-sized genes by conventional techniques. The platform we used, although providing much less information than whole-exome or whole-genome sequencing, has the advantage that can also be run on 'benchtop' sequencers combining rapid turnaround times with higher manageability.

Non-invasive prenatal testing (NIPT) is accurate for fetal sex determination in singleton pregnancies, but its accuracy is not well established in twin pregnancies. Here, we present an accurate sex prediction model to discriminate fetal sex in both dichorionic diamniotic (DCDA) and monochorionic diamniotic/monochorionic monoamniotic (MCDA/MCMA) twin pregnancies. A retrospective analysis was performed using a total of 198 twin pregnancies with documented sex. The prediction was based on a multinomial logistic regression using the normalized frequency of X and Y chromosomes, and fetal fraction estimation. A second-step regression analysis was applied when one or both twins were predicted to be male. The model determines fetal sex with 100% sensitivity and specificity when both twins are female, and with 98% sensitivity and 95% specificity when a male is present. Since sex determination can be clinically important, implementing fetal sex determination in twins will improve overall twin pregnancies management.