Diet metabarcoding of Lasiurus semotus: guano
DNA was extracted using a Qiagen DNA PowerSoil Kit (Qiagen, USA) following the manufacturers protocol (2017), with modifications described in Alberdi et al. 2018 or a DNeasy PowerSoil Pro Kit (Qiagen, USA) following manufacturers protocol (2019). Each sample of extracted DNA obtained from bat guano and extraction blank was duplicated and one additional blank was included in each library preparation as a lab negative control for the library construction step. Target gene sequences were amplified by custom primers which contained the Zeale primers and Illumnia adapters on the 5 and 3 ends. PCR was used to amplify prey DNA amplicons in guano samples plus replicates, as well as two reaction blanks of water which were carried through the entire sequencing process. Amplification success was confirmed with gel electrophoresis. Initial PCR products containing adapters were cleaned with Agencourt AMPure XP beads (Beckman Coulter, USA), and a second round of indexing PCR with a Nextera XT library kit (Illumnia, CA, USA) was used to attached unique combinations of indexes, or MID tags (Illumnia, USA), allowing the prey amplicons to be referenced back to individual bats. Indexed PCR products received an additional round of purification with Agencourt AMPure XP beads, then were quantified using a fluorometer, combined into approximately equimolar pools and quantified on a Bioanalyzer (Agilent Technologies, USA) to verify MID tag additions and calculate final loading concentrations for sequencing. Samples and blanks were duplicated, diluted to 4pM, combined with PhiX control DNA at a ratio of 10% PhiX. Of the 141 guano samples, 34 were sequenced using MiSeq Reagent Kit v2 chemistry 300 cycles of 2 X 175 bp paired-end reads, and 107 using MiSeq Reagent Kit v2 chemistry 500 cycles of 2 x 250 bp paired-end reads.