scholarly journals Single Molecule Sequencing of Cell-free DNA from Maternal Plasma for Noninvasive Trisomy Detection

2017 ◽  
Author(s):  
Minyue Dong ◽  
Liwei Deng ◽  
Huan Jin ◽  
Jinsen Cai ◽  
Huan Shang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Early Stage ◽  
Maternal Plasma ◽  
Trisomy 13 ◽  
Cell Free Dna ◽  
Single Molecule Sequencing ◽  
Fetal Dna ◽  
Free Dna ◽  
Autosomal Aneuploidy ◽  
Cell Free Fetal Dna

AbstractThe demand of non-invasive prenatal testing for autosomal aneuploidy using cell-free fetal DNA (cffDNA) in maternal plasma is a highly sought-after diagnostic, with a rapidly growing market. Current approaches developed by next generation sequencing (NGS) need PCR amplifcation during sample preparation, which results in amplification bias in GC-rich areas of the human genome. With these approaches, the minimum fetal fraction in maternal plasma is 4% for the small differences in circulating cfDNA between trisomic and disomic pregnancies to be detectable. In this paper, we performed single molecule sequencing of cell-free DNA from maternal plasma for noninvasive trisomy 13, 18 and 21 detections using the GenoCare platform. We found that single molecule sequencing is sensitive enough to detect these chromosome abnormalities when the fetal DNA fraction is as low as 2%. Compared to the Hiseq2500 platform, no significant GC bias was observed. The improved sensitivity and unbiased GC readout make GenoCare a promising platform for autosomal aneuploidy detections, even in the very early stage of pregnancy.

scholarly journals Qualitative and quantitative comparison of cell-free DNA and cell-free fetal DNA isolation by four (semi-)automated extraction methods: impact in two clinical applications: chimerism quantification and noninvasive prenatal diagnosis

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Pascal Pedini ◽  
Hajer Graiet ◽  
Laurine Laget ◽  
Lugdivine Filosa ◽  
Jade Chatron ◽  
...  
Keyword(s):  
Fragment Size ◽  
Statistical Tests ◽  
Extraction Methods ◽  
Maternal Plasma ◽  
Isolation Procedure ◽  
Cell Free Dna ◽  
Quantification Method ◽  
Fetal Dna ◽  
Free Dna ◽  
Cell Free Fetal Dna

Abstract Background Non-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In this study, we compared the efficiencies of four (semi)-automated cfDNA isolation instruments using their respective isolation kit: MagNA Pure 24 (Roche®), IDEAL (IDSolution®), LABTurbo 24 (Taigen®) and Chemagic 360 (Perkin Elmer®). The cfDNA was isolated from 5 plasma samples and the Rhesus D (RhD)-cffDNA from 5 maternal plasmas. The cfDNA were quantified by digital droplet PCR (ddPCR), BIABooster system and QUBIT fluorometer. The cfDNA fragment size profiles were assessed by BIABooster system. Chimerism were quantified by home-made ddPCR and Devyser NGS kit. RhD-cffDNA in maternal plasma were detected between weeks 14 and 24 of amenorrhea using free DNA Fetal RHD Kit® (Biorad®). Results Statistical tests have shown differences in DNA yield depending on the isolation procedure and quantification method used. Magna Pure isolates smaller cfDNA fragment size than other extraction methods (90% ± 9% vs. 74% ± 8%; p = 0.009). Chimerism was only reliable from LABTurbo 24 extractions using the NGS but not with ddPCR whatever extraction methods. RhD-cffDNA were detected by all isolation methods, although IDEAL and LABTurbo 24 systems seemed more efficient. Conclusions This comparative study showed a dependency of cfDNA yield depending on isolation procedure and quantification method used. In total, these results suggest that the choice of pre-analytical isolation systems needs to be carefully validated in routine clinical practice.

scholarly journals Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex

2016 ◽  
Vol 62 (6) ◽  
pp. 848-855 ◽  
Author(s):  
George Koumbaris ◽  
Elena Kypri ◽  
Kyriakos Tsangaras ◽  
Achilleas Achilleos ◽  
Petros Mina ◽  
...  
Keyword(s):  
Trisomy 21 ◽  
Dna Analysis ◽  
Prenatal Testing ◽  
Cost Effective ◽  
Maternal Plasma ◽  
Trisomy 13 ◽  
Cell Free Dna ◽  
Free Dna ◽  
Genomic Regions ◽  
Fetal Fraction

Abstract BACKGROUND There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). METHODS We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. RESULTS Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%–100%) cases of trisomy 21, 16/16 (95% CI, 79.4%–100%) cases of trisomy 18, 5/5 (95% CI, 47.8%–100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%–100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%–100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. CONCLUSIONS The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

scholarly journals False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

2016 ◽  
Vol 2016 ◽  
pp. 1-5
Author(s):  
Yang Cao ◽  
Nicole L. Hoppman ◽  
Sarah E. Kerr ◽  
Christopher A. Sattler ◽  
Kristi S. Borowski ◽  
...  
Keyword(s):  
Prenatal Screening ◽  
False Negative ◽  
Chromosome Abnormalities ◽  
Trisomy 13 ◽  
Cell Free Dna ◽  
Free Dna ◽  
Negative Cell ◽  
Cell Free Fetal Dna ◽  
Cfdna Screening ◽  
Screening Result

Background.Noninvasive prenatal screening (NIPS) is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA) circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy.Results.We report a case of false negative cell-free DNA (cfDNA) screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism.Conclusions.Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information.

scholarly journals Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma

2021 ◽  
Vol 118 (50) ◽  
pp. e2114937118
Author(s):  
Stephanie C. Y. Yu ◽  
Peiyong Jiang ◽  
Wenlei Peng ◽  
Suk Hang Cheng ◽  
Y. T. Tommy Cheung ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Analysis ◽  
Large Population ◽  
Maternal Plasma ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Dna Molecule ◽  
Free Dna ◽  
Monogenic Diseases

In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5′ fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5′ 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.

scholarly journals Single Molecule Sequencing of Free DNA from Maternal Plasma for Noninvasive Trisomy 21 Detection

2012 ◽  
Vol 58 (4) ◽  
pp. 699-706 ◽  
Author(s):  
Jessica M E van den Oever ◽  
Sahila Balkassmi ◽  
E Joanne Verweij ◽  
Maarten van Iterson ◽  
Phebe N Adama van Scheltema ◽  
...  
Keyword(s):  
Single Molecule ◽  
Trisomy 21 ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Maternal Plasma ◽  
Single Molecule Sequencing ◽  
Experimental Noise ◽  
Fetal Aneuploidy ◽  
Free Dna ◽  
Gc Bias

Abstract BACKGROUND Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in amplification bias in GC-rich areas of the human genome. To eliminate this bias, and thereby experimental noise, we have used single molecule sequencing as an alternative method. METHODS For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform. RESULTS Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses. CONCLUSIONS This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.

scholarly journals Successful Noninvasive Trisomy 18 Detection Using Single Molecule Sequencing

2013 ◽  
Vol 59 (4) ◽  
pp. 705-709 ◽  
Author(s):  
Jessica ME van den Oever ◽  
Sahila Balkassmi ◽  
Lennart F Johansson ◽  
Phebe N Adama van Scheltema ◽  
Ron F Suijkerbuijk ◽  
...  
Keyword(s):  
Single Molecule ◽  
Trisomy 21 ◽  
Maternal Plasma ◽  
Trisomy 18 ◽  
Diagnostic Sensitivity ◽  
Trisomy 13 ◽  
Noninvasive Detection ◽  
Single Molecule Sequencing ◽  
Gc Bias ◽  
Diagnostic Sensitivity And Specificity

BACKGROUND Noninvasive trisomy 21 detection performed by use of massively parallel sequencing is achievable with high diagnostic sensitivity and low false-positive rates. Detection of fetal trisomy 18 and 13 has been reported as well but seems to be less accurate with the use of this approach. The reduced accuracy can be explained by PCR-introduced guanine-cytosine (GC) bias influencing sequencing data. Previously, we demonstrated that sequence data generated by single molecule sequencing show virtually no GC bias and result in a more pronounced noninvasive detection of fetal trisomy 21. In this study, single molecule sequencing was used for noninvasive detection of trisomy 18 and 13. METHODS Single molecule sequencing was performed on the Helicos platform with free DNA isolated from maternal plasma from 11 weeks of gestation onward (n = 17). Relative sequence tag density ratios were calculated against male control plasma samples and results were compared to those of previous karyotyping. RESULTS All trisomy 18 fetuses were identified correctly with a diagnostic sensitivity and specificity of 100%. However, low diagnostic sensitivity and specificity were observed for fetal trisomy 13 detection. CONCLUSIONS We successfully applied single molecule sequencing in combination with relative sequence tag density calculations for noninvasive trisomy 18 detection using free DNA from maternal plasma. However, noninvasive trisomy 13 detection was not accurate and seemed to be influenced by more than just GC content.

Circulating Maternal Total Cell-Free DNA, Cell-Free Fetal DNA and Soluble Endoglin Levels in Preeclampsia: Predictors of Adverse Fetal Outcome? A Cohort Study

2016 ◽  
Vol 20 (2) ◽  
pp. 135-149 ◽  
Author(s):  
Radwa Marawan AbdelHalim ◽  
Dalia Ibrahim Ramadan ◽  
Reham Zeyada ◽  
Ahmed Soliman Nasr ◽  
Iman Atef Mandour
Keyword(s):  
Cohort Study ◽  
Fetal Outcome ◽  
Total Cell ◽  
Cell Free Dna ◽  
Fetal Dna ◽  
Free Dna ◽  
Soluble Endoglin ◽  
Cell Free Fetal Dna ◽  
Adverse Fetal Outcome

scholarly journals Qualitative and Quantitative Comparison of Cell-Free DNA and Cell-Free Fetal DNA Isolation by Four (Semi-)Automated Extraction Methods: Impact in Two Clinical Applications: Chimerism Quantification and Noninvasive Prenatal Diagnosis

2020 ◽  
Author(s):  
Pedini Pascal ◽  
Hajer GRAIET ◽  
Laurine LAGET ◽  
Lugdivine FILOSA ◽  
Jade CHATRON ◽  
...  
Keyword(s):  
Fragment Size ◽  
Extraction Methods ◽  
Isolation Procedure ◽  
Statistical Testing ◽  
Cell Free Dna ◽  
Quantification Method ◽  
Fetal Dna ◽  
Free Dna ◽  
Cell Free Fetal Dna ◽  
Isolation Methods

Abstract Background: Non-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In this study, we compared the efficiencies of four (semi)-automated cfDNA isolation instruments using their respective isolation kit: MagNA Pure 24 (Roche®), IDEAL (IDSolution®), LABTurbo 24 (Taigen®) and Chemagic 360 (Perkin Elmer®). The cfDNA was isolated from 5 plasma samples and the Rhesus D (RhD)-cffDNA from 5 maternal plasmas. The cfDNA were quantified by digital droplet PCR (ddPCR), BIABooster system and QUBIT fluorometer. The cfDNA fragment size profiles were assessed by BIABooster system. Chimerism were quantified by home-made ddPCR and Devyser NGS kit. RhD-cffDNA in maternal plasma were detected between weeks 14-24 of amenorrhea using free DNA Fetal RHD Kit® (Biorad®). Results:Statistical tests have shown differencies in DNA yield depending on the isolation procedure and quantification method used. Magna Pure isolates smaller cfDNA fragment size than other extraction methods (90% ± 9% vs 74% ± 8%; p=0.009). Chimerism was only reliable from LABTurbo 24 extractions using the NGS but not with ddPCR whatever extraction methods. RhD-cffDNA were detected by all isolation methods, although IDEAL and LABTurbo 24 systems seemed more efficient. Conclusions: 4 (semi-)automated isolation methods have been compared for their extraction efficiency of cfDNA as well as for their fragment size, in a context of chimerism quantification and RHD cffDNA detection. Statistical testing showed a dependency of cfDNA yield on isolation procedure and quantification method used. In total, this study suggests that the choice of pre-analytical isolation systems needs to be carefully validated in routine clinical practice.

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