scholarly journals EP04.30: Detection of fetal low rate of mosaicism Trisomy 8 by next‐generation sequencing of maternal plasma and invasive diagnosis

2019 ◽  
Vol 54 (S1) ◽  
pp. 257-257
Author(s):  
K. Yan
Keyword(s):  
Next Generation Sequencing ◽  
Maternal Plasma ◽  
Next Generation ◽  
Trisomy 8 ◽  
Generation Sequencing ◽  
Low Rate

Towards the Development of a Noninvasive Prenatal Test for Beta-Thalassemia: Utilization of Probe Capture Enrichment and Next Generation Sequencing

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3622-3622 ◽  
Author(s):  
Katie Carlberg ◽  
Nikhil Bose ◽  
Jingyi Deng ◽  
Ashutosh Lal ◽  
Henry Erlich ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Globin Gene ◽  
Maternal Plasma ◽  
Paternal Allele ◽  
Next Generation ◽  
Mixture Ratio ◽  
Minor Fraction ◽  
Cell Free Fetal Dna ◽  
Generation Sequencing ◽  
Fetal Genotype

Abstract β-thalassemia causes significant morbidity and mortality worldwide. Currently, the diagnosis can be made prenatally for couples at risk using invasive procedures such as chorionic villus sampling and amniocentesis. Noninvasive prenatal testing (NIPT) on maternal blood samples would enable earlier fetal diagnosis and eliminate risks associated with these procedures. The discovery of cell-free fetal DNA (cfFDNA) in maternal plasma and advances in next generation sequencing (NGS) have made NIPT a clinical reality for aneuploidies. Diagnosing autosomal recessive (AR) disorders is more challenging as it requires determination of both the paternally and maternally inherited alleles and can be particularly difficult when both parents carry the same mutation. Previous studies used methods to identify the paternally inherited allele in maternal plasma through the detection of a DNA sequence variant present in the father and absent in the mother. Our method expands the target region beyond the β-globin gene into highly polymorphic regions to increase the likelihood of identifying unique paternal sequences. Unlike the detection of the paternal allele, there is no direct qualitative approach for determining the maternally inherited allele. Our solution is an indirect quantitative method that compares the ratio of allelic sequence reads to infer which maternal allele was inherited by the fetus. We have developed a novel NGS assay which utilizes probe capture enrichment, a method employing thousands of short overlapping oligonucleotide probes complementary to a target sequence region. This design makes it uniquely applicable to short, fragmented DNA, such as cell-free DNA (~150 base pairs). Our probe assay targets a contiguous 900 bp region which spans a portion of the β-globin gene (part of exon 2 as well as the entirety of IVS-1 and exon 1) and extends 579 bp into the highly polymorphic 5' UTR region to identify linked sequence variations. Additionally, the assay targets 451 single nucleotide polymorphisms (SNPs) throughout the genome. SNPs that are "informative" (i.e. an allele present in the fetus and absent in the mother) are used to calculate the fraction of cfDNA from the fetus. We have evaluated our assay's performance in a set of experiments designed to simulate the challenging aspects of cfFDNA: very low quantity and short fragment size. The assay's sensitivity was tested by preparing libraries containing amounts of DNA ranging from 50 to 0.05 ngs. At DNA amounts as low as 5 ng (5-fold lower than that expected in plasma), 100% coverage was achieved for the targeted β-globin region and SNPs. The probe/NGS system successfully captured and sequenced DNA fragments as short as 35 bps and as little as 0.5 ng of DNA with >95% coverage. To test the ability of the probe/NGS system to resolve mixtures, DNA samples from patients with known β-globin mutations were combined in ratios ranging from 2.5:97.5 to 20:80 in 25 ng total DNA to mimic the maternal/fetal cfDNA mixture. Our assay was able to detect minority heterozygous mutant alleles at proportions as low as 1.25% and 0.3 ng of DNA. In a mixture designed to simulate a case with a shared parental mutation (CD41/42(-TTCT)), we identified a linked SNP (rs713040) allele, which was within 250 bp of the mutation and unique to the minor fraction. We used this SNP to distinguish the CD41/42 (-TTCT) mutation contributed by the minor fraction. Based on 6 mixtures, we observed 24.3% (110/451) of SNPs to be informative, allowing for a precise estimate of the minor fraction. We estimated the minor fraction to be 11.88% and 5.98% compared to the expected 10% and 5%, respectively. To show proof of concept for inferring the minor ("fetal") genotype when the major ("maternal") genotype was heterozygous mutant (IVS1-5 (G>C)), the estimated minor fraction (10.4% based on 104 informative SNPs) was used to calculate the expected allelic ratios of the 3 different possible minor ("fetal") genotypes. The minor genotype was correctly inferred as wt/wt based on the observed mixture ratio (42.56/57.41 mut/wt) compared to the expected (44.93/55.07). These data show that our probe capture/NGS system can overcome the challenges implicit in the analysis of cfFDNA for NIPT: low DNA amount (<5 ng) and short fragments (<150 bp). We expect that our approach using the fetal fraction estimate will allow us to successfully infer the fetal genotype when applied to maternal plasma. Disclosures Erlich: Allen and Overy, Law Office: Consultancy.

Prediction of fetal blood group and platelet antigens from maternal plasma using next-generation sequencing

Transfusion ◽  
2019 ◽  
Vol 59 (3) ◽  
pp. 1102-1107 ◽  
Author(s):  
Agnieszka Orzińska ◽  
Katarzyna Guz ◽  
Michal Mikula ◽  
Anna Kluska ◽  
Aneta Balabas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Blood Group ◽  
Maternal Plasma ◽  
Next Generation ◽  
Fetal Blood ◽  
Generation Sequencing

The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies

2013 ◽  
Vol 33 (7) ◽  
pp. 667-674 ◽  
Author(s):  
Jacob A. Canick ◽  
Glenn E. Palomaki ◽  
Edward M. Kloza ◽  
Geralyn M. Lambert-Messerlian ◽  
James E. Haddow
Keyword(s):  
Next Generation Sequencing ◽  
Maternal Plasma ◽  
Next Generation ◽  
Plasma Dna ◽  
Fetal Aneuploidies ◽  
The Impact ◽  
Generation Sequencing ◽  
Fetal Fraction

scholarly journals Detection of Microdeletion 22q11.2 in a Fetus by Next-Generation Sequencing of Maternal Plasma

2012 ◽  
Vol 58 (7) ◽  
pp. 1148-1151 ◽  
Author(s):  
Taylor J Jensen ◽  
Zeljko Dzakula ◽  
Cosmin Deciu ◽  
Dirk van den Boom ◽  
Mathias Ehrich
Keyword(s):  
Next Generation Sequencing ◽  
22Q11.2 Deletion Syndrome ◽  
Maternal Plasma ◽  
Deletion Syndrome ◽  
Sequencing Analysis ◽  
Next Generation ◽  
22Q11.2 Deletion ◽  
Fetal Dna ◽  
Chromosome 22Q11.2 ◽  
Generation Sequencing

Abstract BACKGROUND Efforts have been undertaken recently to assess the fetal genome through analysis of circulating cell-free (ccf) fetal DNA obtained from maternal plasma. Sequencing analysis of such ccf DNA has been shown to enable accurate prenatal detection of fetal aneuploidies, including trisomies of chromosomes 21, 18, and 13. We sought to extend these analyses to examine subchromosomal copy number variants through the sequencing of ccf DNA. We examined a clinically relevant genomic region, chromosome 22q11.2, the location of a series of well-characterized deletion anomalies that cause 22q11.2 deletion syndrome. METHODS We sequenced ccf DNA isolated from maternal plasma samples obtained from 2 patients with confirmed 22q11.2 deletion syndrome and from 14 women at low risk for fetal chromosomal abnormalities. The latter samples were used as controls, and the mean genomic coverage was 3.83-fold. Data were aligned to the human genome, repetitive regions were removed, the remaining data were normalized for GC content, and z scores were calculated for the affected region. RESULTS The median fetal DNA contribution for all samples was 18%, with the affected samples containing 17%–18% fetal DNA. Using a technique similar to that used for sequencing-based fetal aneuploidy detection from maternal plasma, we detected a statistically significant loss of representation of a portion of chromosome 22q11.2 in both of the affected fetal samples. No such loss was detected in any of the control samples. CONCLUSIONS Noninvasive prenatal diagnosis of subchromosomal fetal genomic anomalies is feasible with next-generation sequencing.

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