Metabarcoding of bulk arthropod DNA from vane traps
Libraries of each marker were prepared for Illumina sequencing using a two-step PCR approach. No template controls (NTCs) were included alongside eDNA samples during library preparation to monitor for contamination. For PCR1, each primer pair was synthesized with the overhang adapters on the forward and reverse primers described in Illumina (2016), which are compatible with Illumina Nextera indices. PCR1 reactions were set up in triplicate for each eDNA sample consisting of 12.5 l Quantabio qPCR PerfeCTa ToughMix, 9.5 l PCR grade water, 1 l pooled forward and reverse adapter-tailed primer (10 M each stock concentration), and 2 l template DNA. Thermal cycling of the 16S and Bombus amplicons consisted of 98 C for 2 min followed by 40 cycles of 98 C for 20s, 50 C for 30s,72 C for 30 s, ending with a final extension at 72 C for 5 min. Thermal cycling of the COI amplicons was the same except the first five cycles used an annealing temperature of 45 C, followed by 35 cycles with an annealing temperature of 50 C. Triplicate PCR1 reactions for each sample were pooled (75 l), and 25 l of this pooled product was purified with 30 l of Ampure beads following Illumina (2016). Ten base pair long unique dual indices (UDIs) compatible with the overhang adapters were added to each sample in PCR2 following the reaction set up and reagents described in Illumina (2016) protocol with no changes.  Each final purified amplicon was quantified using a Qubit dsDNA High Sensitivity assay. All flower eDNA (i.e., filter halves) amplicons of COI, Bombus, and 16S were pooled and paired-end sequenced at a loading concentration of 6 pM and 10% phiX on an Illumina MiSeq 500 cycle cartridge at USGS EESC-LRL. The vane trap libraries of COI, Bombus, and 16S were sequenced on a separate MiSeq cartridge with the same loading characteristics.