title: 16S rRNA gene amplicon sequencing of honey bee gut
design: We amplified the V4 region of the 16s rRNA gene using primers 515F-Nex (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA) and 806R-Nex (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT), containing the Illumina adapter sequences for Nextera XT indexes and the primers of the V4 region of the 16S rRNA gene (Caporaso et al. 2011), as described in the Illumina 16S metagenomic sequencing library preparation guide (https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf) and by Kenerov et al. (2020). The first PCR step was performed in a total volume of 25 L, using 12.5 L of 2 Phanta Max Master Mix (Vazyme, Nanjing, China), 5l of MilliQ water, 2.5l of each primer (5M), and 2.5l of template DNA. The PCR program consisted of an initial denaturation step at 98C for 30 s, followed by 25 cycles of 98C for 10 s, 55C for 20 s and 72C for 20 s, and a final extension step at 72C for 5 min. Amplifications were verified by 2% agarose gel electrophoresis. PCR products were purified with Clean NGS purification beads in a 1:0.8 ratio of PCR products to beads, and eluted in 27 L Tris (10 mM, pH 8.5). We next performed a second PCR step to append unique dual indexes to each sample. The PCR was performed in a total volume of 25 L, using 12.5 L of 2 Phanta Max Master Mix (Vazyme, Nanjing, China), 5l of MilliQ water, 2.5l of Nextera XT index primers 1 and 2 (Nextera XT Index kit, Illumina) and 2.5l of template DNA. The PCR program consisted of an initial denaturation step at 95C for 3min followed by eight cycles of 95C for 30s, 55C for 30s and 72C for 30s, and a final extension step at 72C for 5min. The final libraries were purified using Clean NGS purification beads in a 1:1.1 ratio of PCR product to beads, and eluted in 27.5l of 10mM Tris, pH 8.5. The amplicon concentrations, including the negative PCR controls, were then quantified by PicoGreen and pooled in equimolar concentrations (with the exception of the negative controls and the blank extractions, which were pooled in equal volumes instead). We verified that the final pool was of the right size using a Fragment Analyzer (Advanced Analytical) and performed sequencing on an Illumina MiSeq sequencer, producing 2250 bp reads, at the Genomic Technology Facility of the University of Lausanne.