Abstract
BackgroundWith the speedy development of sequencing technologies, noninvasive prenatal testing (NIPT) has been widely applied in clinical practice for testing for fetal aneuploidy. The cell-free fetal DNA (cffDNA) concentration in maternal plasma is the most critical parameter for this technology because it affects the accuracy of NIPT-based sequencing for fetal trisomies 21, 18 and 13. Several approaches have been developed to calculate the cffDNA fraction of the total cell-free DNA in the maternal plasma. However, most approaches depend on specific single nucleotide polymorphism (SNP) allele information or are restricted to male fetuses.MethodsIn this study, we present an innovative method to accurately deduce the concentration of the cffDNA fraction using only maternal plasma DNA. SNPs were classified into four maternal-fetal genotype combinations and three boundaries were added to capture effective SNP loci in which the mother was homozygous and the fetus was heterozygous. The median value of the concentration of the fetal DNA fraction was estimated using the effective SNPs. A depth-bias correction was performed using simulated data and corresponding regression equations for adjustments when the depth of the sequencing data was below 100-fold or the cffDNA fraction is less than 10%.ResultsUsing our approach, the median of the relative bias was 0.4% in 18 maternal plasma samples with a median sequencing depth of 125-fold. There was a significant association (r = 0.935) between our estimations and the estimations inferred from the Y chromosome. Furthermore, this approach could precisely estimate a cffDNA fraction as low as 3%, using only maternal plasma DNA at the targeted region with a sequencing depth of 65-fold. We also used PCR instead of parallel sequencing to calculate the cffDNA fraction. There was a significant association (r = 98.2%) between our estimations and those inferred from the Y chromosome.
Highlights
The results showed that a majority of the points were located around the slanting line, indicating that the estimated results were close to those of the standard values except when the cellfree fetal DNA (cffDNA) fraction was lower than 8% (Fig 3)
The results of our study showed that the depth of sequencing and the number of single nucleotide polymorphism (SNP) loci used in the model were two important factors
The accuracy of the cffDNA fraction deduction was affected by the detectability of the minor alleles at each SNP locus, which was critical for the categorization of the maternal–fetal genotype combination
Summary
The discovery of cell-free fetal DNA (cffDNA) in maternal plasma has inspired various noninvasive prenatal testing (NIPT) applications [1], such as genotyping of the fetal RhD blood group [2, 3], fetal sex determination for sex-linked disorders [4], chromosomal aneuploidy detection [5,6,7,8,9,10,11,12,13,14] and monogenic disease detection [15, 16]. The cellfree fetal DNA (cffDNA) concentration in maternal plasma is the most critical parameter for this technology because it affects the accuracy of NIPT-based sequencing for fetal trisomies 21, 18 and 13. Most approaches depend on specific single nucleotide polymorphism (SNP) allele information or are restricted to male fetuses
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