16S rRNA gene amplicon sequencing of Megaponera analis thorax
16S rRNA gene amplicon sequencing data were obtained from 40 experimental samples, a mock sample (to verify consistency of the MiSeq run compared to previous studies in our group), two blank DNA extractions and a negative PCR control with only H2O. We followed the Illumina 16S metagenomic sequencing preparation guide (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf) to amplify and sequence the V4 region of the 16S rRNA gene. Primers for the first PCR step were 515F-Nex (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA) and 806R-Nex (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT). PCR amplifications were performed in a mix of 12.5 L of Invitrogen Platinum SuperFi DNA Polymerase Master Mix, 5 L of MilliQ water, 2.5 L of each primer (5 M), and 2.5 L of template DNA. PCR conditions were 98 C for 30 s, 25 cycles of 98 C for 10 s, 55 C for 20 s, and 72 C for 20 s, and a final extension step at 72 C for 5 min. Amplifications were confirmed by 2% agarose gel electrophoresis. The PCR products were then purified with Clean NGS purification beads (CleanNA) in a 1:0.8 ratio of PCR product to beads, and eluted in 27.5 L of 10 mM Tris, pH 8.5. We then performed a second PCR step in which unique dual-index combinations were appended to the amplicons using the Nextera XT index kit (Illumina). Second-step PCRs were performed in a 25 L, using 2.5 L of the PCR products, 12.5 L of Invitrogen Platinum SuperFi DNA Polymerase Master Mix, 5 L of MilliQ water, and 2.5 L of each of the Nextera XT indexing primers 1 and 2. PCR conditions were 95 C for 3 min followed by eight cycles of 30 s at 95 C, 30 s at 55 C, and 30 s at 72 C, and a final extension step at 72 C for 5 min. The libraries were again purified using Clean NGS purification beads in a 1:1.12 ratio of PCR product to beads, and eluted in 27.5 L of 10 mM Tris, pH 8.5. The amplicon concentrations, including the negative PCR control, mock and blanks, were quantified by PicoGreen and pooled in equimolar concentrations (except for the water and blank samples which were kept at lower concentrations). We verified that the final pool was of the right size using a Fragment Analyzer (Advanced Analytical). MiSeq (Illumina) sequencing was then performed at the Genomic Technology Facility of the University of Lausanne, producing (2  250 bp) reads.