Plasma-based Prenatal DNA Sequencing

Peripheral blood from pregnant women was collected into EDTA tubes and kept at 4 °C until plasma separation. Blood plasma was separated within 36 h after collection and stored at −20 °C at a DNA isolation unit. DNA was isolated using a QIAgen DNA Blood Mini Kit (Hilden, Germany). Standard fragment libraries were prepared from isolated DNA using a modified protocol of the Illumina TruSeq Nano Kit (San Diego, CA, USA) as described previously [20 (link)]. Briefly, to decrease the laboratory costs we used reduced volumes of reagents, which was compensated for by completing 9 cycles of PCR instead of 8, as per protocol. Physical size selection of cfDNA fragments was performed using specific volumes of magnetic beads in order to enrich FF. Illumina NextSeq 500/550 High Output Kit v2 (San Diego, CA, USA) (75 cycles) was used for massively parallel sequencing of prepared libraries using pair-end sequencing with read length of 35 bp.