scholarly journals Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA

2015 ◽  
Vol 112 (47) ◽  
pp. 14670-14675 ◽  
Author(s):  
Ai-hua Yin ◽  
Chun-fang Peng ◽  
Xin Zhao ◽  
Bennett A. Caughey ◽  
Jie-xia Yang ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Genetic Diagnosis ◽  
Maternal Plasma ◽  
Sequencing Depth ◽  
Comparative Genomic ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Sequencing Platform ◽  
Free Dna ◽  
Semiconductor Sequencing

Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.

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 Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

2020 ◽  
Vol 47 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Klaus Rieneck ◽  
Christoffer Egeberg Hother ◽  
Frederik Banch Clausen ◽  
Marianne Antonius Jakobsen ◽  
Thomas Bergholt ◽  
...  
Keyword(s):  
Pregnant Woman ◽  
Next Generation Sequencing ◽  
Pregnant Women ◽  
Blood Group ◽  
Maternal Plasma ◽  
Clinical Samples ◽  
Abo Blood Group ◽  
Cell Free Dna ◽  
Free Dna ◽  
Generation Sequencing

Introduction: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. Objective: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group. Materials and Methods: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. Results: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. Conclusions: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

scholarly journals A New Method for Improving Extraction Efficiency and Purity of Urine and Plasma Cell-Free DNA

Diagnostics ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 650
Author(s):  
Selena Y. Lin ◽  
Yue Luo ◽  
Matthew M. Marshall ◽  
Barbara J. Johnson ◽  
Sung R. Park ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Amplification Efficiency ◽  
Normal Donor ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Pcr Product ◽  
Cleanup Procedure ◽  
Dna Profiles ◽  
The Impact

This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA than did kits M or Q (p < 0.001) from urine, and similar amounts from plasma (p = 0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p < 0.001) and plasma (p ≤ 0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p = 0.05). We demonstrate that DNApure can provide an efficient means of improving the yield and purity of cfDNA and minimize the effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

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