Non-invasive Fetal DNA Analysis from Maternal Blood

Blood from pregnant women was collected into EDTA tubes and kept at 4 °C temperature until plasma separation. Blood plasma was separated within 36 h after collection and stored at −20 °C unit DNA isolation. DNA was isolated using Qiagen DNA Blood Mini kit (Hilden, Germany). Standard fragment libraries for massively parallel sequencing were prepared from isolated DNA using an Illumina TruSeq Nano kit (San Diego, CA, USA) and a modified protocol described previously [15 (link)]. Briefly, to decrease laboratory costs, we used reduced volumes of reagents what were compensated by 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 fetal fraction. 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 2×35bp on an Illummina NextSeq 500 platform (Available online: https://www.illumina.com/).