16S rRNA sequencing of the bumblebee faecal microbiota
DNA extraction from bee gut samples was performed using the Zymo Quick-DNA microprep kit (Cambridge Bioscience, Cambridge, UK) following the manufacturers instructions. Polymerase chain reaction (PCR) was performed using the Roche High-Fidelity PCR System (Roche Life Science, Welwyn Garden City, UK). A total of 5ng DNA was amplified in a reaction volume of 10L. The primers for the amplification of the 16S V3-V4 region were: ACACTGACGACATGGTTCTACACCTACGGGNGGCWGCAG (forward) and TACGGTAGCAGAGACTTGGTCTGACTACHVGGGTATCTAATCC (reverse). The PCR reaction mixture included 1l of 10X FastStart High Fidelity Reaction buffer, 0.1 l of 10 M forward or reverse primers, 1.8L of 25mM MgCl2, 0.5L dimethyl sulfoxide (DMSO), 0.2L of 10mM PCR Grade Nucleotide Mix, 0.1L of 5U/L FastStart High Fidelity Enzyme Blend and 5.2L of nuclease-free water. This was added to 1L of diluted samples and amplified for 35 cycles at 95C for 30s, 55C for 30 s, 72C for 30s, and a final extension at 72C for 5 minutes. The size of amplified products was verified by electrophoresis on a 2% agarose gel. We then used 1 L of a 100 times diluted PCR product in 10mM Tris/HCl, 1mM EDTA pH 7.5 buffer in a second round of PCR to add TSP FLD barcodes and Illumina adaptors onto amplification products. The barcoding reaction mix included 1L of 10X FastStart High Fidelity Reaction buffer, 1.8L of 25mM MgCl2, 0.5L of DMSO, 0.2L of 10mM PCR Grade Nucleotide Mix, 0.1L of 5U/L FastStart High Fidelity Enzyme Blend and 3.4L of nuclease-free water. This was added to 2L of Fluidigm Barcode and 1L of the 1:100 harvested PCR product. The amplification was done for 15 cycles at 95C for 15s, 60C for 30 s, 72C for 60s, and a final extension at 72C for 3 minutes. Barcode attachment was controlled using the Tapestation D1000 instrument (Agilent, CA, USA).  An equal volume of each barcoded PCR product was pooled, and the final mixture diluted to 4nM.  The pooled library was loaded onto a 300bpx2 paired-end MiSeq (Illumina, CA, USA), as per the manufacturers instructions.