Miseq 2 250 v2 kit

PCR amplicons for the V4 region of the 16S rRNA gene were generated with primers 515F–806R^{63 (link)} and were sequenced with the Illumina MiSeq 2 × 250 v2 Kit at the Cornell University Institute for Biotechnology. DADA2^{27 (link)} was used to call 100% sequence identity OTUs (i.e., sequence variants). Taxonomy was assigned to OTUs with the QIIME2 q2-feature-classifier^{64 (link)} using the SILVA database (v119)^{65 (link)}. The phyloseq^{66 (link)} R package was used to rarefy total OTU counts to 5000 per sample due to the multiple orders of magnitude difference in raw counts among samples. A phylogeny was inferred for all OTU sequences with fasttree^{67 (link)} based on a multiple sequence alignment generated by mafft^{68 (link)}. All samples lacking metadata used in the study were filtered from the dataset. In cases where an individual host was sampled multiple times, we randomly selected one sample.

PCR was performed using universal primers flanking the variable 4 (V4) region of the bacterial 16 S rRNA gene44 (link). A total of 50 ng DNA, 0.4 μM each primer, 12.5 μl 2X HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA), and water to 25 μl were used for one reaction per sample. Cycling conditions were as follows: initial denaturation of 95 °C for 3 min followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 5 min. PCR products were purified by gel extraction from a 1.0% low-melt agarose gel (National Diagnostics, Atlanta, GA) using a ZR-96 Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). Samples were quantified by Qubit^® Fluorometer and equimolar pooled. The pool plus 5% PhiX control DNA was sequenced with the MiSeq 2 × 250 v2 kit (Illumina, San Diego, CA, USA) using custom sequencing primers44 (link).

PCR was performed using universal primers flanking the variable 4 (V4)
region of the bacterial 16S rRNA gene^{39 (link)}. Genomic DNA samples were amplified in duplicate. Each
reaction contained 25 ng genomic DNA, 10 μM of each uniquely barcoded
primer, 12.5 μl 2x HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington,
MA, USA), and water to a final reaction volume of 25 μl. PCR was carried
out under the following conditions: initial denaturation for 3 min at
95°C, followed by 20 cycles of denaturation for 30 s at 95°C,
annealing for 30 s at 55°C and elongation for 30 s at 72°C, and a
final elongation step for 5 min at 72°C. PCR products were purified with
the QIAquick 96-well PCR Purification Kit and quantified using the Qubit dsDNA
HS Assay kit (Invitrogen, Oregon, USA). Samples were equimolar pooled and
sequenced by the University of Wisconsin–Madison Biotechnology Center
with the MiSeq 2×250 v2 kit (Illumina, San Diego, CA, USA) using custom
sequencing primers.