16S rRNA Amplicon Library Preparation

Amplicon libraries were prepared as described by the Illumina protocol [25 ]. Briefly, a PCR of 25 cycles was performed to amplify the V3–V4 region of the 16S rRNA gene prior to the incorporation of the Illumina overhang adapters (Second PCR: 10 cycles) using 2.5 µL of DNA sample (5 ng/µL), 0.5 µL of each primer, and 12.5 µL of 2x KAPA HiFi Hotstart ReadyMix (Kapa Biosystems, Merck, Darmstadt, Germany). Bacterial communities were assessed using universal primers (Bac341F: CCT ACG GGN GGC WGC AG and Bac805R: GAC TAC HVG GGT ATC TAA TCC) [26 (link)] covering the V3–V4 regions of the 16S rRNA gene. The third PCR amplification (8 cycles) was carried out using Nextera XT index primers (Illumina, San Diego, CA, USA). Amplifications were conducted on a Veriti^® 96-Well Thermal Cycler (Applied Biosystems^®, Thermo Scientific, Waltham, MA, USA). The PCR products were purified with AMPure XP beads and Veriti^® 96-Well Thermal Cycler (Applied Biosystems^®, Thermo Scientific, Waltham, MA, USA), as described in the protocol. Amplicon libraries were then sequenced on the Illumina MiSeq (Illumina, San Diego, CA, USA), generating 2 × 300 bp paired-end reads.