Noninvasive Detection of Fetal Genetic Variations through Polymorphic Sites Sequencing of Maternal Plasma DNA

Non-invasive prenatal testing (NIPT) for common fetal aneuploidies using circulating cell free DNA in maternal plasma has been widely adopted in clinical practice for its sensitivity and accuracy. However, the detection of subchromosomal abnormalities or monogenetic variations showed no cost-effectiveness or satisfactory accuracy. Here we developed an assay, the goodness-of-fit and graphical analysis of polymorphic sites based non-invasive prenatal testing (GGAP-NIPT), to simultaneously detect fetal chromosomal/subchromosomal and nucleotide level abnormalities. In each sample, fetal fraction was estimated using allelic counts of reference polymorphic sites and a robust linear regression model. Then the genotype of each polymorphic site was estimated using allelic goodness of fit test. Finally, monogenic mutations were detected using allelic wildtype and mutant counts of each target site, and chromosomal/subchromosomal abnormalities were identified by collective analysis of all target polymorphic sites. Such an analytic approach was highly accurate for detecting aneuploidies, microdeletions or microduplications and monogenic mutations for simulated samples with different fetal fractions and sequencing depths. Moreover, more than 93% of fetal monogenic mutations were correctly identified for target hotspot sites amplified using circulating or barcode-enabled single-molecule assays. With the aid of sample replicates, higher detection accuracy was observed. Through target polymorphic sites sequencing, all chromosomal/subchromosomal and monogenic abnormalities could be detected simultaneously, facilitating the extension of NIPT to an expanded panel of genetic disorders in a cost-effective manner.