Abstract
Non-invasive prenatal testing (NIPT) for aneuploidy on Chromosomes 21 (T21), 18 (T18) and 13 (T13) is actively used in clinical practice around the world. One of the limitations of the wider implementation of this test is the high cost of the analysis itself, as high-throughput sequencing is still relatively expensive. At the same time, there is an increasing trend in the length of reads yielded by sequencers. Since extracellular DNA is short, in the order of 140–160 bp, it is not possible to effectively use long reads. The authors used high-performance sequencing of cell-free DNA (cfDNA) libraries that went through additional stages of enzymatic fragmentation and random ligation of the resulting products to create long chimeric reads. The authors used a controlled set of samples to analyze a set of cfDNA samples from pregnant women with a high risk of fetus aneuploidy according to the results of the first trimester screening and confirmed by invasive karyotyping of the fetus using laboratory and analytical approaches developed by the authors. They evaluated the sensitivity, specificity, PPV (positive predictive value), and NPV (negative predictive value) of the results. The authors developed a technique for constructing long chimeric reads from short cfDNA fragments and validated the test using a control set of extracellular DNA samples obtained from pregnant women. The obtained sensitivity and specificity parameters of the NIPT developed by the authors corresponded to the approaches proposed earlier (99.93% and 99.14% for T21; 100% and 98.34% for T18; 100% and 99.17% for T13, respectively).
Highlights
Non-invasive prenatal testing (NIPT) studies are carried out early in the pregnancy to determine the presence of aneuploidy in the fetal genome
The goal of this work was to expand the laboratory and bioinformatics stages of NIPT using analysis of tags in chimeric molecules built from cell-free DNA (cfDNA) fragments of pregnant women (Figure 1)
Insertions, deletions, chromosomes X and Y, mitochondrial DNA and the p-arm of chromosome 6 (HLA region) were not included; Only diallelic polymorphisms were considered; Only SNP with identifiers “rs” were considered; Only alleles with a minimum allele frequency (MAF) of 0.4 were considered; We considered SNPs with a minimum probability of selection according to Hardy–Weinberg of p
Summary
NIPT (non-invasive prenatal testing) studies are carried out early in the pregnancy 9–12 weeks of gestation) to determine the presence of aneuploidy in the fetal genome. This test is done using freely circulating cell-free DNA (cfDNA) isolated from the mother’s blood, part of which is of placental origin [1]. Cell-free fetal DNA (cffDNA) enters the mother’s blood as a result of apoptosis of placental trophoblast cells and reaches a proportion of 4–15% by the end of the first trimester of pregnancy [2,3]. In the mother’s blood, cfDNA is in the form of fragments. Fragments of maternal cfDNA are predominantly 166 bp in length, and cffDNA fragments are predominantly. This distribution of the lengths is associated with non-random DNA cutting [5]
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