Spri beads

Libraries were generated using barcoded PCR primers 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 338R 5′-TGCTGCCTCCCGTAGGAGT-3′ annealing to the V1–V2 region of the 16S rRNA gene^{36 (link)}. PCR reactions were carried out in quadruplicate using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA). After amplification, quadruplicate PCR reactions were pooled and then purified using a 1:1 volume of SPRI beads (GE HealthCare, Chicago, IL). DNA in each sample was then quantified using Quant-iT PicoGreen Assay Kit (Thermo Fisher) and pooled in equal molar amounts. The resulting library was sequenced on the MiSeq instrument (Illumina, San Diego, CA) using 2 × 250 bp chemistry. Extraction blanks and DNA-free water were subjected to the same amplification and purification procedure to allow for empirical assessment of environmental and reagent contamination. Positive controls, consisting of eight artificial 16S gene fragments synthesized in gene blocks and combined in known abundances, were also included^{37 (link)}.

Bacterial DNA was extracted as described previously [43 (link)] using the PureLink kit (K182002, Invitrogen) and quantified using the Quant-iT PicoGreen Assay Kit (P7589, Invitrogen). The 16S ribosomal RNA genes were amplified using bar-coded PCR primers targeted to the V1-V3 hypervariable region (FP 5′-AGAGTTTGATCCTGGCTCAG-3′; RC 5-ATTACCGCGGCTGCTGG-3′). PCR reactions were carried out in quadruplicate using Accuprime Taq HiFi (12346086, Invitrogen). Each PCR reaction contained 0.2 µM of each primer, 1 U Accuprime Taq HiFi, 1× Buffer II, and 2 µL DNA in a total volume of 25 µL. Cycling conditions are as follows: 1 cycle of 95 °C for 2 min; 32 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 60 s; 1 cycle of 72 °C for 5 min. The resulting 16S rDNA amplicons were purified using a 1:1 volume of SPRI beads (09-981-123, GE Healthcare, Chicago, IL, USA), quantified using PicoGreen, pooled in equal amounts, and sequenced on the Illumina MiSeq using 2 × 300 bp chemistry. Extraction blanks and DNA-free water were subjected to the same amplification and purification procedure to assess potential environmental contamination. Library preparation and sequencing were performed at the CHOP Microbiome Center (University of Pennsylvania, Philadelphia, PA, USA).