• Humans
• Female
• Pregnancy
• Cell-Free Nucleic Acids/blood
• Cell-Free Nucleic Acids/analysis
• DNA Methylation
• Noninvasive Prenatal Testing/methods
• Polymerase Chain Reaction/methods
• Male
• Fetus/metabolism
• Prospective Studies
• Prenatal Diagnosis/methods
• Reproducibility of Results

• R01 HD098258 NICHD NIH HHS
• 1R01HD098258 Eunice Kennedy Shriver National Institute of Child Health and Human Development
• Department of Pediatrics, NYU Langone Health
• Bio-Rad Laboratories Inc.
• Eunice Kennedy Shriver National Institute of Child Health and Human Development

• Humans
• Pregnancy
• Placenta/metabolism
• Female
• Cell-Free Nucleic Acids/genetics
• Cell-Free Nucleic Acids/blood
• Noninvasive Prenatal Testing/methods

• Female
• Child
• Humans
• Pregnancy
• Paternity
• Polymorphism, Single Nucleotide
• Fetus
• High-Throughput Nucleotide Sequencing/methods
• Genotype
• Cell-Free Nucleic Acids

• cell-free fetal DNA
• fetal fraction
• genetic counseling
• molecular genetics
• noninvasive prenatal screening

• amplicon sequencing
• fetal fraction
• goodness of fit
• noninvasive prenatal testing
• polymorphic site
• robust linear regression

• NIPT
• low cell-free DNA (cfDNA) fetal fraction
• miRNA

• Fluorescence
• Nanobiosensor
• SRY gene
• Sex determination
• cffDNA
• r-GQD@HTAB

The molecular pathology of hemolytic disease of the fetus and newborn (HDFN) is determined by different RHD, RHCE, and KEL genotypes and by blood group incompatibility between the mother and fetus that is caused by erythrocyte antigen presence/absence on the cell surface. In the Czech Republic, clinically significant antierythrocyte alloantibodies include anti-D, anti-K, anti C/c, and anti-E. Deletion of the RHD gene and then three single nucleotide polymorphisms in the RHCE and KEL genes (rs676785, rs609320, and rs8176058) are the most common. The aim of this study is to develop effective and precise monitoring of fetal genotypes from maternal plasma of these polymorphisms using droplet digital (dd)PCR. Fifty-three plasma DNA samples (from 10 to 18 weeks of gestation) were analyzed (10 RHD, 33 RHCE, and 10 KEL). The ddPCR methodology was validated on the basis of the already elaborated and established method of minisequencing and real-time PCR and with newborn phenotype confirmation. The results of ddPCR were in 100% agreement with minisequencing and real-time PCR and also with newborn phenotype. ddPCR can fully replace the reliable but more time-consuming method of minisequencing and real-time PCR RHD examination. Accurate and rapid noninvasive fetal genotyping minimizes the possibility of HDFN developing.

• KEL
• RHCE
• RHD
• blood group incompatibility
• cell-free fetal DNA
• droplet digital PCR
• hemolytic disease of fetus and newborn
• noninvasive fetal genotyping

• biological techniques
• genetics
• biomarkers
• molecular medicine

• Adult
• Cell-Free Nucleic Acids/analysis
• Cell-Free Nucleic Acids/genetics
• Chromosomes, Human, X/genetics
• Female
• Fetus/chemistry
• Fetus/metabolism
• Genetic Testing
• Humans
• INDEL Mutation
• Polymerase Chain Reaction/methods
• Polymorphism, Genetic
• Pregnancy
• Prenatal Diagnosis/methods
• Sex Determination Analysis/methods

• Ministry of Education, Youth and Sport of the Czech Republic
• Ministry of Health of the Czech Republic
• Ministry of Education, Youth and Sport of the Czech Republic,

• Cell-free DNA (cfDNA)
• DNA mixtures
• Massively parallel sequencing (MPS)
• Microhaps
• Noninvasive prenatal paternity testing (NIPPT)
• Single nucleotide polymorphisms (SNPs)

To review the performance of noninvasive prenatal screening (NIPS) using targeted single‐nucleotide polymorphisms (SNPs) approach in mixed‐risk Thai women.

Retrospective analysis of data for detection of trisomy 21 (T21), 18 (T18), 13 (T13), monosomy X (XO), other sex chromosome aneuploidies (SCA), and triploidy/vanishing twins (VT) from a single commercial laboratory.

Mean (±SD) gestational age and maternal weight were 13.2 (±2.1) weeks and 125.7 (±22.4) pounds, respectively (n = 8,572). From 462/8,572 (5.4%) no‐calls; 1/462 (0.2%) was uninformative SNPs, and 1/462 chose amniocentesis. Redraw settled 323/460 (70%) samples with low fetal fraction (FF); and 8,434/8,572 (98.4%) were finally reportable, with 131 high risks (1.6%). The median (min‐max) FF of reportable (n = 8,434) and unreportable samples (n = 137) samples were 10.5% (2.6–37.9) and 3.8% (1–14.1), respectively (p < .05). Fetal karyotypes were available in 106/131 (80.9%) and 52/138 (37.7%) high risk and repeated no‐calls, respectively. The positive predictive values (PPVs) for T21 (n = 47), T18 (n = 15), T13 (n = 7), XO (n = 8), other SCA (n = 7), and triploidy/VT were 94%, 100%, 58.3%, 66.7%, 70%, and 57.1%, respectively. None of repeated no‐calls had aneuploidies.

SNP‐based NIPS has high PPVs for T21 and T18. Although the proprietary SNPs library is not population‐specific, uninformative SNPs are uncommon.

• DNA-based screening
• Down syndrome
• autosomal trisomy
• noninvasive prenatal screening
• sex chromosomal aneuploidies
• single nucleotide polymorphisms

Echinococcosis is a life-threatening parasitic disease caused by Echinococcus spp. tapeworms with over one million people affected globally at any time. The Echinococcus spp. tapeworms in the human body release DNA to the circulatory system, which can be a biomarker for echinococcosis. Cell-free DNA (cfDNA) is widely used in medical research and has been applied in various clinical settings. As for echinococcosis, several PCR-based tests had been trialed to detect cell-free Echinococcus spp. DNA in plasma or serum, but the sensitivity was about 20% to 25%. Low sensitivity of PCR-based methods might be related to our limited understanding of the features of cell-free Echinococcus spp. DNA in plasma, including its concentration, fragment pattern and release source. In this study, we applied ultra-high-throughput sequencing to comprehensively investigate the characteristics of cell-free Echinococcus spp. DNA in plasma of echinococcosis patients.

We collected plasma samples from 23 echinococcosis patients. Total plasma cfDNA was extracted and sequenced with a high-throughput sequencing platform. An average of 282 million read pairs were obtained for each plasma sample. Sequencing data were analyzed with bioinformatics workflow combined with Echinococcus spp. sequence database. After identification of cell-free Echinococcus spp. reads, we found that the cell-free Echinococcus spp. reads accounted for 1.8e-5 to 4.0e-9 of the total clean reads. Comparing fragment length distribution of cfDNA between Echinococcus spp. and humans showed that cell-free Echinococcus spp. DNA of cystic echinococcosis (CE) had a broad length range, while that of alveolar echinococcosis (AE) had an obvious peak at about 135 bp. We found that most of the cell-free Echinococcus spp. DNA reads were from the nuclear genome with an even distribution, which might indicate a random release pattern of cell-free Echinococcus spp. DNA.

With ultra-high-throughput sequencing technology, we analyzed the concentration, fragment length, release source, and other characteristics of cell-free Echinococcus spp. DNA in the plasma of echinococcosis patients. A better understanding of the characteristics of cell-free Echinococcus spp. DNA in plasma may facilitate their future application as a biomarker for diagnosis.

Echinococcosis is one of the most neglected tropical diseases caused by the metacestodes of Echinococcus spp. tapeworms, which affect both humans and livestock. Plasma cell-free DNA (cfDNA) consists of nucleic acid fragments found extracellularly and may contain DNA released from the parasites. Research shows that a variety of parasites can be detected from plasma cfDNA. Cell-free Echinococcus spp. DNA in plasma or serum had been tested with PCR-based methods, but these PCR methods had low sensitivity ranged from 20% to 25%. Low sensitivity may be due to our limited understanding of cell-free Echinococcus spp. DNA in plasma. Here, we take advantage of high-throughput sequencing to get a comprehensive characterization of cell-free Echinococcus spp. DNA. Our results showed that with high-throughput sequencing we could detect cell-free Echinococcus spp. DNA in all samples, though at a very low level. Based on the sequencing data, we found that cell-free Echinococcus spp. DNA in plasma had a different fragment length distribution to cell-free human DNA, and fragment length distribution of cell-free Echinococcus spp. DNA is also different between cystic echinococcosis (CE) and alveolar echinococcosis (AE). The sequencing data can also help trace the release source of cell-free Echinococcus spp. DNA from the genome. According to the mapping results of cell-free Echinococcus spp. DNA reads, we found that most of them were from the nuclear genome rather than the mitochondrial genome, and their release position showed an even distribution on the genome. These characteristics of cell-free Echinococcus spp. DNA in echinococcosis patients’ plasma could facilitate their future application in research or clinical settings.

• Alloimmune thrombocytopenia
• Cell-free (fetal) DNA
• Fetus
• Hemolytic disease
• Newborn
• Next-generation sequencing
• Noninvasive fetal blood group genotyping

Non‐invasive prenatal testing (NIPT) for fetal aneuploidies has rapidly been incorporated into clinical practice. Current NGS‐based methods can reliably detect fetal aneuploidies non‐invasively with fetal fraction of at least 4%. Inaccurate fetal fraction assessment can compromise the accuracy of the test as affected samples with low fetal fraction have an increased risk for misdiagnosis. Using a novel set of fetal‐specific differentially methylated regions (DMRs) and methylation sensitive restriction digestion (MSRD), we developed a multiplex ddPCR assay for accurate detection of fetal fraction in maternal plasma.

We initially performed MSRD followed by methylation DNA immunoprecipitation (MeDIP) and NGS on fetal and non‐pregnant female tissues to identify fetal‐specific DMRs. DMRs with the highest methylation difference between the two tissues were selected for fetal fraction estimation employing MSRD and multiplex ddPCR. Chromosome Y multiplex ddPCR assay (YMM) was used as a reference standard, to develop our fetal fraction estimation model in male pregnancy samples. Additional 123 samples were tested to examine whether the model is sex dependent and/or ploidy dependent.

In all, 93 DMRs were identified of which seven were selected for fetal fraction estimation. Statistical analysis resulted in the final model which included four DMRs (FFMM). High correlation with YMM‐based fetal fractions was observed using 85 male pregnancies (r = 0.86 95% CI: 0.80–0.91). The model was confirmed using an independent set of 53 male pregnancies.

By employing a set of well‐characterized DMRs, we developed a SNP‐, sex‐ and ploidy‐independent methylation‐based multiplex ddPCR assay for accurate fetal fraction estimation.

• differentially methylated regions
• fetal fraction estimation
• multiplex ddPCR
• non-invasive prenatal testing

• Cell-free DNA
• Non-invasive
• PGT-A
• Preimplantation genetic screening
• Spent embryo media
• cfDNA