Identification of fetal unmodified and 5-hydroxymethylated CGs in maternal cell-free DNA for non-invasive prenatal testing
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ABSTRACT:
PROVIDER: PRJNA626634 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11575400.fastq.gz | Fastqsanger.gz | |||
| SRR11575401.fastq.gz | Fastqsanger.gz | |||
| SRR11575402.fastq.gz | Fastqsanger.gz | |||
| SRR11575403.fastq.gz | Fastqsanger.gz | |||
| SRR11575404.fastq.gz | Fastqsanger.gz |
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Project description:Massively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test.
2020-10-20 | GSE148964 | GEO
Project description:BackgroundMassively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test.MethodsWe employed uTOPseq and hmTOP-seq approaches which combine covalent derivatization of unmodified or hydroxymethylated CG sites, respectively, with next generation sequencing, or quantitative real-time PCR.ResultsWe detected increased 5-hydroxymethylcytosine (5hmC) levels in fetal chorionic villi (CV) tissue samples as compared with peripheral blood. Using our previously developed uTOP-seq and hmTOP-seq approaches we obtained whole-genome uCG and 5hmCG maps of 10 CV tissue and 38 cfDNA samples in total. Our results indicated that, in contrast to conventional whole genome sequencing, such epigenomic analysis highly specifically enriches fetal DNA fragments from maternal cfDNA. While both our approaches yielded 100% accuracy in detecting Down syndrome in fetuses, hmTOP-seq maintained such accuracy at ultra-low sequencing depths using only one million reads. We identified 2164 and 1589 placenta-specific differentially modified and 5-hydroxymethylated regions, respectively, in chromosome 21, as well as 3490 and 2002 Down syndrome-specific differentially modified and 5-hydroxymethylated regions, respectively, that can be used as biomarkers for identification of Down syndrome or other epigenetic diseases of a fetus.ConclusionsuTOP-seq and hmTOP-seq approaches provide a cost-efficient and sensitive epigenetic analysis of fetal abnormalities in maternal cfDNA. The results demonstrated that T21 fetuses contain a perturbed epigenome and also indicated that fetal cfDNA might originate from fetal tissues other than placental chorionic villi. Robust covalent derivatization followed by targeted analysis of fetal DNA by sequencing or qPCR presents an attractive strategy that could help achieve superior sensitivity and specificity in prenatal diagnostics.
| S-EPMC7574562 | biostudies-literature
Project description:Whereas non-invasive prenatal testing for aneuploidies (NIPT-A) is widely implemented, non-invasive prenatal testing for monogenic diseases (NIPT-M) is lagging. By capturing and targeted sequencing of 250000 polymorphic SNP loci from maternal plasma circulating cell-free DNA (cfDNA) and DNA from relatives, the fetal haplotype and chromosomal copy numbers are deduced. In all families tested, the cfDNA derived haplotypes are on average 97% concordant with the neonatal and embryo haplotype. This generic non-invasive prenatal diagnostic approach allows cost efficient scrutinizing the fetal genome for the presence of any inherited monogenic disease or trait.
| EGAS00001003634 | EGA
Project description:BACKGROUND:Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening. METHODS:Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110) and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104). RESULTS:The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10 °C to 28 °C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10-39, n = 1317). CONCLUSION:The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.
| S-EPMC3800077 | biostudies-literature
Project description:Current non-invasive prenatal screening (NIPS) analyzes circulating fetal cell-free DNA (cfDNA) in maternal peripheral blood for selected aneuploidies or microdeletion/duplication syndromes. Many genetic disorders are refractory to NIPS largely because the maternal genetic material constitutes most of the total cfDNA present in the maternal plasma, which hinders the detection of fetus-specific genetic variants. Here, we developed an innovative sequencing method, termed coordinative allele-aware target enrichment sequencing (COATE-seq), followed by multidimensional genomic analyses of sequencing read depth, allelic fraction, and linked single nucleotide polymorphisms, to accurately separate the fetal genome from the maternal background. Analytical confounders including multiple gestations, maternal copy number variations, and absence of heterozygosity were successfully recognized and precluded for fetal variant analyses. In addition, fetus-specific genomic characteristics, including the cfDNA fragment length, meiotic error origins, meiotic recombination, and recombination breakpoints were identified which reinforced the fetal variant assessment. In 1129 qualified pregnancies tested, 54 fetal aneuploidies, 8 microdeletions/microduplications, and 8 monogenic variants were detected with 100% sensitivity and 99.3% specificity. Using the comprehensive cfDNA genomic analysis tools developed, we found that 60.3% of aneuploidy samples had aberrant meiotic recombination providing important insights into the mechanism underlying meiotic nondisjunctions. Altogether, we show that the genetic deconvolution of the fetal and maternal cfDNA enables thorough and accurate delineation of fetal genome which paves the way for the next-generation prenatal screening of essentially all types of human genetic disorders.
| S-EPMC9562363 | biostudies-literature
Project description:Objectives: To assess the efficacy of Y-chromosome mini-STR-based next-generation sequencing (NGS) for non-invasive prenatal paternity testing (NIPPT). Methods: DNA was extracted from the plasma of 24 pregnant women, and cell-free fetal DNA (cffDNA) genotyping was performed at 12 Y-chromosome mini-STR loci using the Illumina NextSeq 500 system. The cffDNA haplotype was validated by the paternal haplotype. The paternity testing parameters were attributed to each case quantitatively. Results: The biological relationship between the alleged fathers and infants in all 24 family cases were confirmed by capillary electrophoresis (CE). The Y-chromosome mini-STR haplotypes of all 14 male cffDNA were obtained by NGS without any missing loci. The alleles of cffDNA and paternal genomic DNA were matched in 13 cases, and a mismatched allele was detected at the DYS393 locus in one case and considered as mutation. No allele was detected in the 10 female cffDNA. The combined paternity index (CPI) and probability of paternity calculation was based on 6 loci Y-haplotype distributions of a local population. The probability of paternity was 98.2699-99.8828% for the cases without mutation, and 14.8719% for the case harboring mutation. Conclusions: Our proof-of-concept study demonstrated that Y-chromosome mini-STR can be used for NGS-based NIPPT with high accuracy in real cases, and is a promising tool for familial searching, paternity exclusion and sex selection in forensic and medical applications.
2021-10-25 | GSE186434 | GEO
Project description: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.
| S-EPMC6609680 | biostudies-other
Project description:Background:Non-invasive prenatal testing (NIPT) has been confirmed as the most accurate screening test for trisomies 21, 18, and 13. However, reports on NIPT performance in sex chromosome aneuploidies (SCA) based on real clinical data are still limited. Methods:High-throughput massively parallel genomic sequencing (MPS) technique was used to screen for fetal SCAs as part of the research to determine the potential value of NIPT in detecting fetal SCAs in the second trimester. A number of 12,243 consecutive cases from a single center were included in this study. Results:The positive predictive value (PPV) of NIPT in the present study was 57.6%, which was divided and categorized by individual SCAs as follows: 21.4% for Turner syndrome (45,X), 75.0% for Triple X syndrome (47,XXX), 90.9% for Klinefelter syndrome (47,XXY), and 75.0% for XYY syndrome (47,XYY). Conclusion:The NIPT-based SCA test cannot be used as a diagnostic method, and performing an invasive confirmation test on NIPT-based SCA-positive cases is strongly recommended.
| S-EPMC7068885 | biostudies-literature
Project description:We report a new case of 46,XX male syndrome that was detected following an anomalous result by non-invasive prenatal testing (NIPT) and a discrepancy between the fetal karyotype and the ultrasonographic investigation. With the increasing use of NIPT, more gender discordances can be identified prenatally and be amenable to early therapy.
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Project description:Non-invasive prenatal testing (NIPT) has become a routine practice in screening for common aneuploidies of chromosomes 21, 18, and 13 and gonosomes X and Y in fetuses worldwide since 2015 and has even expanded to include smaller subchromosomal events. In fact, the fetal fraction represents only a small proportion of cell-free DNA on a predominant background of maternal DNA. Unlike fetal findings that have to be confirmed using invasive testing, it has been well documented that NIPT provides information on maternal mosaicism, occult malignancies, and hidden health conditions due to copy number variations (CNVs) with diagnostic resolution. Although large duplications or deletions associated with certain medical conditions or syndromes are usually well recognized and easy to interpret, very little is known about small, relatively common copy number variations on the order of a few hundred kilobases and their potential impact on human health. We analyzed data from 6422 NIPT patient samples with a CNV detection resolution of 200 kb for the maternal genome and identified 942 distinct CNVs; 328 occurred repeatedly. We defined them as multiple occurring variants (MOVs). We scrutinized the most common ones, compared them with frequencies in the gnomAD SVs v2.1, dbVar, and DGV population databases, and analyzed them with an emphasis on genomic content and potential association with specific phenotypes.
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