Identification of fetal unmodified and 5-hydroxymethylated CGs in maternal cell-free DNA for non-invasive prenatal testing

Massively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test. We employed covalent derivatization of CG sites combined with lower cost next generation sequencing, or quantitative PCR for analysis of fetal chorionic villi tissue samples, and cfDNA of pregnant and non-pregnant women.