Nextera xt index

Bacterial genomic DNA was extracted from the calf feces (487 samples from 63 calves), maternal milk (six samples), and feed pellets (two samples) using a Repeated Bead-Beating plus column (QIAamp DNA stool mini kit; Qiagen, Valencia, CA, USA)^{61 (link)} (Supplementary Tables 1–4). In preparation for Illumina MiSeq sequencing, a fragment of the 16 S rRNA gene spanning the hypervariable V3–V4 region was amplified by PCR using the forward primer 338F (5′-TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3′) and the reverse primer 805R (5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′). PCR was performed in a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: initial denaturation at 95 °C for 3 min; followed by 23 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s; and then a final extension step at 72 °C for 5 min. Products of three PCR reactions using the same template were pooled and purified using the QIAquick PCR purification kit (Qiagen). For the Illumina MiSeq sequencing, a 16S V3–V4 PCR product library was prepared using the Nextera XT Index (Illumina). The library was then sequenced on the Illumina MiSeq platform and a paired-end 2 × 300 bp reagent kit, according to the manufacturer’s instructions.

The DNA concentration of the samples was adjusted to 10 ng/μl and then diluted at 1:20 for the subsequent investigations. The sequencing protocol was performed at BMR Genomics Srl (Padua, Italy). Briefly, the V3-V4 regions of 16S rRNA gene were amplified using the following primers: Pro341F, 5′-CCTACGGGNBGCASCAG-3′, and Pro805R, 5′-GACTACN VGGGTATCTAATCC-3′ (Takahashi et al., 2014 (link)). Primers were modified with the forward and reverse overhangs (5′-TCGT CGGCAGCGTCAGATGTGTATAAGAGACAG-[locus-specific sequence]-3′ and 5′-GTCTCGTGGGCTCGGAGATGTGTAT AAGAGACAG-[locus-specific sequence]-3′, respectively) necessary for dual index library preparation. Amplicons were purified by 0.8x Agencourt AMPure XP magnetic beads (Beckman Coulter) and amplified with a short cycle with a Nextera XT Index (Illumina). They were then normalized by SequalPrep (Thermo Fisher) and multiplexed. The pool was purified by 1x Agencourt AMPure XP magnetic beads (Beckman Coulter), loaded on Illumina Miseq, and sequenced with a 300PE v3 chemistry strategy.