16S rRNA sequencing of Pogonomyrmex ant internal bacterial communities
Library preparation and sequencing were performed by Novogene at Novogenes Nanjing Sequencing Center (Nanjing, China). 16S rRNA genes of distinct region 16SV4 were amplified used specific primers 515F (Parada et al. 2016) and 806R (Apprill et al. 2015). All PCR reactions were carried out with 15 L of Phusion High-Fidelity PCR Master Mix, 0.2 M of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98  for 1 min, followed by 30 cycles of denaturation at 98  for 10 s, annealing at 50 for 30 s, and elongation at 72 for 30 s and 72 for 5 min. The PCR products were purified using magnetic bead purification. Samples were mixed in equidensity ratios based on the concentration of PCR products. After thorough mixing, the PCR products were detected and target bands were recovered. Sequencing libraries were generated and indexes were added. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced, according to effective library concentration and data amount required. Sequencing was performed on an Illumina NovaSeq PE250 sequencing platform