16S rRNA metabarcoding of Crotophaga ani: gut (1h postmortem)
Total genomic DNA (gDNA) was isolated from 15 mg of each gut sample with ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research, Irvine, CA, United States) according to the manufacturer's protocol, quantified with a Qubit Fluorometer (Invitrogen, Life Technologies, CA, USA) and stored immediately at -20°C in the Sacha Wiwakuna Biobank at the Instituto de Investigacion en Zoonosis (CIZ) of the Universidad Central del Ecuador. To identify bacterial communities, a single amplicon of approximately 550 bp, corresponding to the V3-V4 hypervariable region of the prokaryotic 16S rRNA gene, was amplified with the forward primer 341F and the reverse primer 805R. The resultant PCR products were purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA). Library preparation was performed using dual-index barcodes from the Nextera XT Index Kit (Illumina Inc., San Diego, CA, USA).The libraries were purified, quantified and sequencing on Illumina MiSeq platform with paired-end reads of 300 bp using MiSeq reagent kit v3 (600 cycles PE) (Illumina Inc., San Diego, CA, USA).