Powersoil pro

For feces, cryovials containing thawed fecal suspensions were centrifuged at 13,000 RPM, the supernatant was removed, and fecal material was transferred to a fresh microfuge tube. DNA was extracted using the PowerSoil kit following the manufacturer’s instructions (QIAGEN, Germantown, MD), eluted with 100 μl of the provided elution solution, and stored in microfuge tubes at −80°C.
For DNA extraction from breastmilk, preliminary experiments were performed to evaluate DNA isolation methods with respect to quantity of microbial DNA recovered (Supplementary Table S6). Three DNA isolation kits were compared (PowerSoil, PowerFood, and PowerSoil Pro (all available from QIAGEN)) by qPCR using fungal (UNI1 and UNI2 (Heisel et al., 2015 (link))) and bacterial-specific (515f and 806r (Caporaso et al., 2012 (link))) primers. DNA yield was greatest when using the following process: Breastmilk (1 ml) was transferred to a sterile microfuge tube and centrifuged at 13,000 RPM. The supernatant and fat layer were removed, the pellet was resuspended in Solution 1 from the PowerSoil Pro kit, and this solution was then transferred to the first tube in the PowerSoil Pro kit. DNA extraction then proceeded following the manufacturer’s protocol. DNA was eluted with 100 μl of the provided elution solution and was stored in microfuge tubes at −80°C.

For bacterial DNA extraction 700μL of SL1 lysis buffer (NucleoSpin Soil kit, Macherey-Nagel) was added to the stool samples and tubes were heated at 95°C for 2h to inactivate SARS-CoV-2. Samples were then homogenized using the FastPrep-24TM instrument (MP Biomedicals) and extraction was pursued using the NucleoSpin Soil kit according to the manufacturer’s instructions. DNA concentration was assessed using a NanoDrop spectrophotometer. Samples with too low DNA concentration were excluded. DNA from human samples was extracted with PowerSoil Pro (Qiagen) on the QiaCube HT (Qiagen), using Powerbead Pro (Qiagen) plates with 0.5mm and 0.1mm ceramic beads. For mouse samples, the variable region 4 (V4) of the 16S rRNA gene was amplified by PCR using primers containing adapters for MiSeq sequencing and single-index barcodes. All PCR products were analyzed with the Agilent TapeStation for quality control and then pooled equimolar and sequenced directly in the Illumina MiSeq platform using the 2×250 bp protocol. Human samples were prepared with a protocol derived from ^{44 (link)}, using KAPA HiFi Polymerase to amplify the V4 region of the 16S rRNA gene. Libraries were sequenced on an Illumina MiSeq using paired-end 2×250 reads and the MiSeq Reagent Kitv2.

Samples were extracted with PowerSoil Pro (Qiagen) automated for high throughput on the QiaCube HT (Qiagen), using Powerbead Pro Plates (Qiagen) with 0.5 mm and 0.1 mm ceramic beads.