Metabarcoding of Insectivorous Bat Diets

DNA was extracted from feces using the QIAamp DNA stool extraction kit (Qiagen, Valencia, CA) following the modifications described in Zeale et al. (2011) . A short (157 bp excluding primer) region of the cytochrome c oxidase subunit I (COI) mitochondrial DNA gene was subsequently PCR amplified from each DNA extract. We used generic arthropod primers with a wide taxonomic coverage that includes 13 insect and arachnid orders commonly found in insectivorous bat diets (ZBJ-ArtF1c and ZBJ-ArtR2c; Zeale et al. 2011 ). The primers were modified into 5′-tagged “fusion primers,” in order to enable Roche FLX sequencing of pools of the amplicons, and subsequent bioinformatic sorting into original PCR (Binladen et al. 2007 ). Post-PCR amplification, the amplicons were purified, quantified by real-time PCR (qPCR) (Meyer et al. 2007 (link)), and then pooled by species at equimolar ratio. Each pool of amplicons was deep sequenced on one-eighth of a Roche GS-FLX platform using Titanium sequencing chemistry. PCR reaction conditions, cycle program, and sequencing procedures followed Bohmann et al. (2011) (link) and are provided in Supporting Information 2.