scholarly journals A novel high-throughput molecular counting method with single base-pair resolution enables accurate single-gene NIPT

2019 ◽  
Author(s):  
David S. Tsao ◽  
Sukrit Silas ◽  
Brian P. Landry ◽  
Nelda Itzep ◽  
Amy B. Nguyen ◽  
...  
Keyword(s):  
Muscular Atrophy ◽  
Single Gene ◽  
Prenatal Testing ◽  
Maternal Blood ◽  
Beta Thalassemia ◽  
Analytical Sensitivity ◽  
Dna Molecules ◽  
Sequencing Data ◽  
Non Invasive ◽  
Single Base Pair

ABSTRACTNext-generation DNA sequencing is currently limited by an inability to count the number of input DNA molecules. Molecular counting is particularly needed when accurate quantification is required for diagnostic purposes, such as in single-gene non-invasive prenatal testing (sgNIPT) and liquid biopsy. We developed Quantitative Counting Template (QCT) molecular counting for reconstructing the number of input DNA molecules using sequencing data. We then used QCT molecular counting to develop sgNIPT of sickle cell disease, cystic fibrosis, spinal muscular atrophy, alpha-thalassemia, and beta-thalassemia. Incorporating molecular count information into a statistical model of disease likelihood led to analytical sensitivity and specificity of >98% and >99%, respectively. Validation of sgNIPT was further performed with maternal blood samples collected during pregnancy, and sgNIPT was 100% concordant with newborn follow-up.

scholarly journals A novel high-throughput molecular counting method with single base-pair resolution enables accurate single-gene NIPT

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
David S. Tsao ◽  
Sukrit Silas ◽  
Brian P. Landry ◽  
Nelda P. Itzep ◽  
Amy B. Nguyen ◽  
...  
Keyword(s):  
Muscular Atrophy ◽  
Single Gene ◽  
Prenatal Testing ◽  
Maternal Blood ◽  
Beta Thalassemia ◽  
Analytical Sensitivity ◽  
Dna Molecules ◽  
Sequencing Data ◽  
Non Invasive ◽  
Single Base Pair

Abstract Next-generation DNA sequencing is currently limited by an inability to accurately count the number of input DNA molecules. Molecular counting is particularly needed when accurate quantification is required for diagnostic purposes, such as in single gene non-invasive prenatal testing (sgNIPT) and liquid biopsy. We developed Quantitative Counting Template (QCT) molecular counting to reconstruct the number of input DNA molecules using sequencing data. We then used QCT molecular counting to develop sgNIPTs of sickle cell disease, cystic fibrosis, spinal muscular atrophy, alpha-thalassemia, and beta-thalassemia. The analytical sensitivity and specificity of sgNIPT was >98% and >99%, respectively. Validation of sgNIPTs was further performed with maternal blood samples collected during pregnancy, and sgNIPTs were 100% concordant with newborn follow-up.

scholarly journals A Novel Next-Generation Sequencing Based Assay for Non-Invasive Prenatal Testing of Sickle Cell Disease without Paternal DNA

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2369-2369
Author(s):  
Nelda Itzep ◽  
Celeste K Kanne ◽  
David Tsao ◽  
Oguzhan Atay ◽  
Vivien A Sheehan
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Prenatal Testing ◽  
Maternal Blood ◽  
Beta Thalassemia ◽  
Cell Disease ◽  
Advisory Committees ◽  
Non Invasive ◽  
The Us ◽  
Allele Fraction

Abstract Introduction: Over 300,000 infants are born with sickle cell disease (SCD) every year worldwide, including at least 1,000 in the US. Prenatal diagnosis by amniocentesis or chorionic villus sampling is available; but high cost, invasiveness, and risk of miscarriage limit their use. Recently, non-invasive prenatal testing (NIPT) has become commonplace for aneuploidies, including Trisomy 21. These non-invasive tests operate by genetic analysis of the cell-free fetal DNA (cffDNA) present in maternal blood. The safety and accuracy of NIPT have been key drivers for its rapid and widespread adoption. Yet, no NIPT for SCD or other hemoglobinopathies have been commercialized to date, despite the large numbers of patients affected in the US and worldwide. While de novo mutations can only be of fetal origin and can be identified by available next-generation sequencing (NGS) methods, NIPT for recessively inherited disorders is more challenging. This is because a mother who is a carrier for a recessive disorder contributes a high level of background pathogenic DNA molecules. Therefore, a key technical challenge of NIPT for recessive disorders is developing an assay sensitive enough to detect <5% deviation from 50% allele fraction. To overcome this challenge, we have developed and optimized an NIPT for SCD by assessing the relative mutation dosage of fetal SCD and beta-thalassemia DNA through a novel molecular counting strategy using NGS. Objectives: The primary objective of this study is to evaluate the performance of a novel NIPT for sickle cell disease. Methods: The SCD NIPT assay and associated custom bioinformatics analysis were performed on cfDNA obtained from a training cohort of non-pregnant compound heterozygotes for SCD. The SCD NIPT assay was then performed on a validation cohort of pregnant women with either SCD or sickle cell trait (SCT). The accuracy of the SCD NIPT was evaluated by comparison with newborn screening results. Results: Non-pregnant individuals with genotype HbSE, HbSC, or HbS/beta-thalassemia were included as a training cohort to establish the precision and accuracy of the assay for measuring HbS allele fraction from cfDNA. As expected, the HbS allele fraction in these individuals was 0.500 (standard deviation = 0.011, n = 26), and there was no detectable fetal fraction in these samples. Both training and validation cohort results matched the theoretical limit of detection set by the number of cell-free HBB DNA molecules in plasma. The precision and accuracy of the HBB assay on cfDNA were then used in conjunction with >1000 pre-clinical samples (mixtures of sheared SCT and SCD genomic DNA) to determine analytical sensitivity >98% and specificity >99%, even in the absence of paternal DNA. Conclusion: We have developed an assay for non-invasive prenatal testing of sickle cell disease. The results obtained to date indicate that the assay reliably detects fetal SCD when the fetal fraction is as low as 5%, the same limit as aneuploidy NIPT. A fetus with SCD has already been identified, and follow-up is ongoing with >20 pregnancies. Since the HBB NIPT is highly targeted, sequencing cost is <$30 per sample. The ability to ascertain fetal SCD status based only on maternal blood will be valuable in clinical settings where the father is unavailable or sample collection would be inconvenient or time-consuming. Several Phase I/II and Phase III trials for curing SCD or beta-thalassemia using autologous gene-editing of stem cells are currently in progress. SCD NIPT could be particularly useful for deciding to bank umbilical cord blood as a source of stem cells for future gene-editing cures. Disclosures Tsao: BillionToOne: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Atay:BillionToOne: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Effect of preexamination conditions in a centralized-testing model of non-invasive prenatal screening

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Chad Fibke ◽  
Sylvie Giroux ◽  
André Caron ◽  
Elizabeth Starks ◽  
Jeremy D.K. Parker ◽  
...  
Keyword(s):  
Gestational Age ◽  
Prenatal Testing ◽  
Positive Association ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Maternal Body Mass Index ◽  
Sequencing Data ◽  
Non Invasive ◽  
Tube Type ◽  
Sequencing Platforms

Abstract Objectives Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF. Methods A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF. Results We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at −80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy. Conclusions Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.

Non-invasive prenatal testing for fetal aneuploidy by massively parallel DNA sequencing of maternal plasma: the future has arrived today/Nicht-invasiver Pränatal-Test auf fetale Aneuploidie mittels massiv-paralleler DNA-Sequenzanalyse im mütterlichen Plasma: in der Zukunft angekommen

2012 ◽  
Vol 36 (5) ◽  
Author(s):  
Amy Swanson ◽  
Christin Coffeen ◽  
Amy J. Sehnert
Keyword(s):  
Massively Parallel Sequencing ◽  
Prenatal Testing ◽  
The United States ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Massively Parallel ◽  
Clinical Implementation ◽  
Maternal Cell ◽  
Non Invasive ◽  
Fetal Aneuploidy

AbstractAfter decades of research, non-invasive prenatal testing (NIPT) using maternal blood to determine fetal chromosome status has found its way from the research laboratory into clinical practice, triggering a long-awaited paradigm shift in prenatal care. A variety of methods using sequencing of maternal cell-free DNA (cfDNA) have now been studied, primarily demonstrating their ability to detect the most common fetal aneuploidy, trisomy 21 (T21). The focus of this article is on massively parallel sequencing (MPS) with optimized sequence tag mapping and chromosome quantification, which accurately detects T21 as well as multiple other aneuploidies across the genome. The power of this technique resides in its high precision and reduction of variation within and between sequencing runs. Using MPS, classification of aneuploidy status for a given sample can be reliably assigned from the genetic information alone without the need to factor in other maternal pre-test risk or other clinical variables. Performance of this method has been prospectively demonstrated in a rigorous, blinded, multi-center study in the United States. The findings suggest that MPS can be incorporated into existing prenatal screening algorithms to reduce unnecessary invasive procedures. This technology and key considerations for clinical implementation are discussed.

Isolation and whole genome sequencing of fetal cells from maternal blood towards the ultimate non-invasive prenatal testing

2017 ◽  
Vol 37 (13) ◽  
pp. 1311-1321 ◽  
Author(s):  
Fang Chen ◽  
Ping Liu ◽  
Ying Gu ◽  
Zhu Zhu ◽  
Amulya Nanisetti ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Prenatal Testing ◽  
Maternal Blood ◽  
Whole Genome ◽  
Fetal Cells ◽  
Non Invasive

Using chromosomal and single-gene non-invasive prenatal testing to identify the cause of congenital heart defects

2021 ◽  
Vol 132 ◽  
pp. S319-S320
Author(s):  
Mark Hajjar ◽  
Pooja Mohan ◽  
Jacob Wulff ◽  
Melissa Maisenbacher ◽  
J. Dianne Keen-Kim ◽  
...  
Keyword(s):  
Congenital Heart Defects ◽  
Congenital Heart ◽  
Heart Defects ◽  
Single Gene ◽  
Prenatal Testing ◽  
Non Invasive

scholarly journals Performance of cell-free DNA sequencing-based non-invasive prenatal testing: experience on 36,456 singleton and multiple pregnancies

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Marco La Verde ◽  
Luigia De Falco ◽  
Annalaura Torella ◽  
Giovanni Savarese ◽  
Pasquale Savarese ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Retrospective Analysis ◽  
False Negative ◽  
Screening Method ◽  
Prenatal Testing ◽  
Maternal Blood ◽  
Cell Free Dna ◽  
Detection Rates ◽  
Non Invasive ◽  
Free Dna

Abstract Background This paper describes the clinical practice and performance of cell-free DNA sequencing-based non-invasive prenatal testing (NIPT) as a screening method for fetal trisomy 21, 18, and 13 (T21, T18, and T13) and sex chromosome aneuploidies (SCA) in a general Italian pregnancy population. Methods The AMES-accredited laboratory offers NIPT in maternal blood as a screening test for fetal T21, T18, T13 and SCA. Samples were sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay. Results A retrospective analysis was performed on 36,456 consecutive maternal blood samples, including 35,650 singleton pregnancies, 800 twin pregnancies, and 6 triplet pregnancies. Samples were tested between April 2017 and September 2019. The cohort included 46% elevated-risk and 54% low-risk patients. A result indicative of a classic trisomy was found in 356 (1%) of singleton or twin samples: 254 T21, 69 T18, and 33 T13. In addition, 145 results (0.4%) were indicative of a SCA. Of the combined 501 screen-positive cases, 484 had confirmatory diagnostic testing. NIPT results were confirmed in 99.2% (247/249) of T21 cases, 91.2% (62/68) of T18 cases, 84.4% (27/32) of T13 cases, and 86.7% (117/135) of SCA cases. In the 35,955 cases reported as unaffected by a classic trisomy or SCA, no false negative cases were reported. Assuming that false negative results would be reported, the sensitivity of NIPT was 100.00% for T21 (95% Cl 98.47–100.0), T18 (95% Cl 94.17–100.0), and T13 (95% Cl 87.54–100.0). The specificities were 99.99% (95% Cl 99.98–100.0), 99.98% (95% Cl 99.96–100.0), 99.99% (95% Cl 99.97–100.0), and 99.95% (95% Cl 99.92–99.97) for T21, T18, T13, and SCA, respectively. Conclusion This retrospective analysis of a large cohort of consecutive patients who had whole-genome sequencing-based NIPT for classic trisomies and SCA shows excellent detection rates and low false positive rates.

scholarly journals Would I have Wanted to Know? A Qualitative Exploration of Women’s Attitudes, Beliefs and Concerns about Non-Invasive Prenatal Testing for de novo Genetic Conditions after having a Child with a de novo Genetic Disorder

OBM Genetics ◽  
2021 ◽  
Vol 05 (04) ◽  
pp. 1-1
Author(s):  
Sarah Long ◽  
◽  
Roanna Lobo ◽  
Peter O'Leary ◽  
Jan E. Dickinson ◽  
...  
Keyword(s):  
Personal Growth ◽  
De Novo ◽  
Genetic Disorder ◽  
Single Gene ◽  
Genetic Diagnosis ◽  
Prenatal Testing ◽  
Phenomenological Approach ◽  
Population Screening ◽  
Chromosomal Disorders ◽  
Non Invasive

Non-invasive prenatal testing (NIPT) for a panel of 25 single gene disorders became available in Western Australia in 2020 and potentially may be able to test for panels of hundreds of disorders as is the case with reproductive carrier screening. How this information would be used by parents in a population screening model is unknown. We used a phenomenological approach to explore retrospectively whether mothers of children with single gene or chromosomal disorders would have wanted to know about their child’s genetic diagnosis prior to delivery. Themes were identified such as having a child with a de novo disorder and effect on pregnancy outcomes in hypothetical situations, impact on family function, the diagnostic journey and personal growth. These themes related to both the concept of expanded NIPT (ENIPT) and the situation of having a child with a de novo genetic disorder that could now hypothetically be detected through ENIPT. Opinions were divided about whether participants would have wanted to know about their affected child’s condition, indicating any expanded NIPT testing panels would need to be offered in the context of an appropriate comprehensive counselling program. How this would be provided on a population screening level and the role of genetic counselling needs further exploration.

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