scholarly journals Noninvasive Prenatal Diagnosis of Monogenic Diseases by Targeted Massively Parallel Sequencing of Maternal Plasma: Application to β-Thalassemia

2012 ◽  
Vol 58 (10) ◽  
pp. 1467-1475 ◽  
Author(s):  
Kwan-Wood G Lam ◽  
Peiyong Jiang ◽  
Gary J W Liao ◽  
K C Allen Chan ◽  
Tak Y Leung ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Massively Parallel Sequencing ◽  
Globin Gene ◽  
Maternal Plasma ◽  
Massively Parallel ◽  
Sequencing Data ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Noninvasive Prenatal Diagnosis ◽  
Monogenic Diseases

Abstract BACKGROUND A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of maternal plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of maternal plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS Targeted sequencing of maternal plasma DNA for NIPD of monogenic diseases is feasible.

scholarly journals Maternal Plasma DNA Analysis with Massively Parallel Sequencing by Ligation for Noninvasive Prenatal Diagnosis of Trisomy 21

2010 ◽  
Vol 56 (3) ◽  
pp. 459-463 ◽  
Author(s):  
Rossa WK Chiu ◽  
Hao Sun ◽  
Ranjit Akolekar ◽  
Christopher Clouser ◽  
Clarence Lee ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Trisomy 21 ◽  
Massively Parallel Sequencing ◽  
Maternal Plasma ◽  
Chromosome 21 ◽  
Massively Parallel ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Noninvasive Prenatal Diagnosis ◽  
Sequencing By Synthesis

Abstract Background: Noninvasive prenatal diagnosis of trisomy 21 (T21) has recently been shown to be achievable by massively parallel sequencing of maternal plasma on a sequencing-by-synthesis platform. The quantification of several other human chromosomes, including chromosomes 18 and 13, has been shown to be less precise, however, with quantitative biases related to the chromosomal GC content. Methods: Maternal plasma DNA from 10 euploid and 5 T21 pregnancies was sequenced with a sequencing-by-ligation approach. We calculated the genomic representations (GRs) of sequenced reads from each chromosome and their associated measurement CVs and compared the GRs of chromosome 21 (chr21) for the euploid and T21 pregnancies. Results: We obtained a median of 12 × 106 unique reads (21% of the total reads) per sample. The GRs deviated from those expected for some chromosomes but in a manner different from that previously reported for the sequencing-by-synthesis approach. Measurements of the GRs for chromosomes 18 and 13 were less precise than for chr21. z Scores of the GR of chr21 were increased in the T21 pregnancies, compared with the euploid pregnancies. Conclusions: Massively parallel sequencing-by-ligation of maternal plasma DNA was effective in identifying T21 fetuses noninvasively. The quantitative biases observed among the GRs of certain chromosomes were more likely based on analytical factors than biological factors. Further research is needed to enhance the precision for measuring for the representations of chromosomes 18 and 13.

scholarly journals Noninvasive prenatal diagnosis of monogenic disorders based on direct haplotype phasing through targeted linked-read sequencing

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Chao Chen ◽  
Min Chen ◽  
Yaping Zhu ◽  
Lu Jiang ◽  
Jia Li ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Massively Parallel Sequencing ◽  
Effective Means ◽  
Massively Parallel ◽  
Nucleotide Polymorphisms ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Monogenic Disorders ◽  
Noninvasive Prenatal Diagnosis ◽  
Hmw Dna

Abstract Background Though massively parallel sequencing has been widely applied to noninvasive prenatal screen for common trisomy, the clinical use of massively parallel sequencing to noninvasive prenatal diagnose monogenic disorders is limited. This study was to develop a method for directly determining paternal haplotypes for noninvasive prenatal diagnosis of monogenic disorders without requiring proband’s samples. Methods The study recruited 40 families at high risk for autosomal recessive diseases. The targeted linked-read sequencing was performed on high molecular weight (HMW) DNA of parents using customized probes designed to capture targeted genes and single-nucleotide polymorphisms (SNPs) distributed within 1Mb flanking region of targeted genes. Plasma DNA from pregnant mothers also underwent targeted sequencing using the same probes to determine fetal haplotypes according to parental haplotypes. The results were further confirmed by invasive prenatal diagnosis. Results Seventy-eight parental haplotypes of targeted gene were successfully determined by targeted linked-read sequencing. The predicted fetal inheritance of variant was correctly deduced in 38 families in which the variants had been confirmed by invasive prenatal diagnosis. Two families were determined to be no-call. Conclusions Targeted linked-read sequencing method demonstrated to be an effective means to phase personal haplotype for noninvasive prenatal diagnosis of monogenic disorders.

scholarly journals Non-invasive prenatal diagnosis by massively parallel sequencing of maternal plasma DNA

Open Biology ◽  
2012 ◽  
Vol 2 (6) ◽  
pp. 120086 ◽  
Author(s):  
Yuk Ming Dennis Lo
Keyword(s):  
Prenatal Diagnosis ◽  
Massively Parallel Sequencing ◽  
Maternal Plasma ◽  
Massively Parallel ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Non Invasive ◽  
Prenatal Detection ◽  
A Genome ◽  
Chromosomal Aneuploidies

The presence of foetal DNA in the plasma of pregnant women has opened up new possibilities for non-invasive prenatal diagnosis. The use of circulating foetal DNA for the non-invasive prenatal detection of foetal chromosomal aneuploidies is challenging as foetal DNA represents a minor fraction of maternal plasma DNA. In 2007, it was shown that single molecule counting methods would allow the detection of the presence of a trisomic foetus, as long as enough molecules were counted. With the advent of massively parallel sequencing, millions or billions of DNA molecules can be readily counted. Using massively parallel sequencing, foetal trisomies 21, 13 and 18 have been detected from maternal plasma. Recently, large-scale clinical studies have validated the robustness of this approach for the prenatal detection of foetal chromosomal aneuploidies. A proof-of-concept study has also shown that a genome-wide genetic and mutational map of a foetus can be constructed from the maternal plasma DNA sequencing data. These developments suggest that the analysis of foetal DNA in maternal plasma would play an increasingly important role in future obstetrics practice. It is thus a priority that the ethical, social and legal issues regarding this technology be systematically studied.

scholarly journals Noninvasive Prenatal Diagnosis of Duchenne Muscular Dystrophy: Comprehensive Genetic Diagnosis in Carrier, Proband, and Fetus

2015 ◽  
Vol 61 (6) ◽  
pp. 829-837 ◽  
Author(s):  
Seong-Keun Yoo ◽  
Byung Chan Lim ◽  
Jiyoung Byeun ◽  
Hee Hwang ◽  
Ki Joong Kim ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Recombination Event ◽  
Massively Parallel Sequencing ◽  
Genetic Diagnosis ◽  
Maternal Plasma ◽  
Massively Parallel ◽  
Carrier Status ◽  
Parallel Sequencing ◽  
Sequencing Platform ◽  
Noninvasive Prenatal Diagnosis

Abstract BACKGROUND Noninvasive prenatal diagnosis of monogenic disorders using maternal plasma and targeted massively parallel sequencing is being investigated actively. We previously demonstrated that comprehensive genetic diagnosis of a Duchenne muscular dystrophy (DMD) patient is feasible using a single targeted sequencing platform. Here we demonstrate the applicability of this approach to carrier detection and noninvasive prenatal diagnosis. METHODS Custom solution-based target enrichment was designed to cover the entire dystrophin (DMD) gene region. Targeted massively parallel sequencing was performed using genomic DNA from 4 mother and proband pairs to test whether carrier status could be detected reliably. Maternal plasma DNA at varying gestational weeks was collected from the same families and sequenced using the same targeted platform to predict the inheritance of the DMD mutation by their fetus. Overrepresentation of an inherited allele was determined by comparing the allele fraction of 2 phased haplotypes after examining and correcting for the recombination event. RESULTS The carrier status of deletion/duplication and point mutations was detected reliably through using a single targeted massively parallel sequencing platform. Whether the fetus had inherited the DMD mutation was predicted correctly in all 4 families as early as 6 weeks and 5 days of gestation. In one of these, detection of the recombination event and reconstruction of the phased haplotype produced a correct diagnosis. CONCLUSIONS Noninvasive prenatal diagnosis of DMD is feasible using a single targeted massively parallel sequencing platform with tiling design.

scholarly journals Targeted Massively Parallel Sequencing of Maternal Plasma DNA Permits Efficient and Unbiased Detection of Fetal Alleles

2011 ◽  
Vol 57 (1) ◽  
pp. 92-101 ◽  
Author(s):  
Gary JW Liao ◽  
Fiona MF Lun ◽  
Yama WL Zheng ◽  
KC Allen Chan ◽  
Tak Y Leung ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Massively Parallel Sequencing ◽  
Maternal Plasma ◽  
Targeted Sequencing ◽  
Massively Parallel ◽  
Target Enrichment ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Fetal Dna ◽  
Genomic Regions

BACKGROUND Massively parallel sequencing has recently been used in noninvasive prenatal diagnosis. The current costs of this technology are still relatively expensive, however, and sample throughput is still relatively low when it is used as a molecular diagnostic tool. Rather than nonselectively sequencing the genome, target enrichment provides a logical approach for more efficient and cost-effective massively parallel sequencing because it increases the proportion of informative data from the targeted region(s). Existing applications of targeted sequencing have mainly been qualitative analyses of genomic DNA. In this study, we investigated its applicability in enriching selected genomic regions from plasma DNA and the quantitative performance of this approach. METHODS DNA was extracted from plasma samples collected from 12 pregnant women carrying female fetuses. The SureSelect Target Enrichment System (Agilent Technologies) was used to enrich for exons on chromosome X. Plasma DNA libraries with and without target enrichment were analyzed by massively parallel sequencing. Genomic DNA samples of the mother and fetus for each case were genotyped by microarray. RESULTS For the regions targeted by the enrichment kit, the mean sequence coverage of the enriched samples was 213-fold higher than that of the nonenriched samples. Maternal and fetal DNA molecules were enriched evenly. After target enrichment, the coverage of fetus-specific alleles within the targeted region increased from 3.5% to 95.9%. CONCLUSIONS Targeted sequencing of maternal plasma DNA permits efficient and unbiased detection of fetal alleles at genomic regions of interest and is a powerful method for measuring the proportion of fetal DNA in a maternal plasma sample.

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