16S and ITS-seq of honey bee gut
The fungal ITS region (ITS4 and ITS3) and the hyper variable region 4 (V4) of the 16S rDNA gene were amplified by PCR (Next Generation Sequencing Core Facility Colorado State University, Fort Collins, CO, USA). Bacterial 16S rDNA and fungal ITS2 regions were used for amplification to determine microbiome community abundances and taxonomic classifications (Yun et al., 2014, 2018, Zeng et al. 2019). DNA-free samples that verified lack of contamination in the buffer and primer sets were included as negative controls. ZymoBIOMICS Microbial Community Standard (Zymo Research Corporation, Irvine, CA, USA. Product D6305) was a positive control. The universal primers 515F and 806R tailed with Illumina barcoded adapters were used for PCR and heterogeneity spacers were added to increase diversity. Adapters were added after the first PCR cycle. Bacterial 16S and fungal ITS2 PCR amplifications from the pooled gut samples, were successfully performed on all but three individual samples (sample #60, 64, and 78). Samples were pooled to equimolar concentrations and cleaned using 0.6X AMPure XP Bead Clean (Beckman Coulter Life Sciences, Indianapolis, IN). Libraries were pooled at equimolar concentrations and sequenced on an Illumina MiSeq at the aforementioned sequencing core facility, and paired-end, 2 x 250 reads and index reads for multiplexing were generated.