Metabarcoding of Agapostemon virescens
DNA was extracted via a non-destructive method using the Qiagen Blood & Tissue kit as described in Martoni et al. (2021). Following overnight incubation at 56C in 200 uL ATL butter and 20 uL proteinase K, DNA extraction was conducted according to manufacturer instructions. We employed amplicon sequencing techniques in triplicate prior to combining replicates to characterize bacterial, fungal and plant communities using 16S rRNA, ITS and rbcL, respectively. For 16S rRNA, we sequenced the V5-V6 region, using the 799bF-CS1 forward primer (MGGATTAGATACCCKGG) and the 1115R-CS2 reverse primer (AGGGTTGCGCTCGTTG). To sequence ITS, we used the forward primer of ITS1F (CTTGGTCATTTAGAGGAAGTAA) and reverse primer of ITS2 (GCTGCGTTCTTCATCGATGC). For plant metabarcoding, we used the RBCL7 forward primer (CTCCTGAMTAYGAAACCAAAGA) and the RBCL8 reverse primer (GTAGCAGCGCCCTTTGTAAC). All primers were described in McFrederick and Rehan (2016). PCR amplification, library preparation and sequencing were performed at the Centre for Biodiversity Genomics at the University of Guelph, in Guelph, Canada. Adapter removal and demultiplexing was also performed at the sequencing facility.