Extraction of neonatal stool DNA
DNA was extracted from thawed stool samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). For all extractions 290-310 mg of stool was transferred into Lysing Matrix E 2mL tubes (MP Biomedicals, Illkirch-Graffenstaden, France). Tubes without stool were used as negative controls for each batch of samples. Samples underwent 4 cycles of bead beating for 30 seconds using the FastPrep-24 5G Instrument (MP Biomedicals). The suspensions were heated to 95 degrees for 5 minutes and then centrifuged (2 minutes, 12000 rpm). The remaining extraction steps were performed according to the manufacturer’s instructions. DNA was eluted using 100 μL of elution buffer and samples were centrifuged for 2 minutes to elute the DNA. DNA yield was assessed using the Qubit dsDNA HS Assay kit with a Qubit 2.0 fluorometer (Invitrogen). Extracted DNA was stored at -80 degrees until amplified.