ITS2 DNA metabarcoding of strongyle parasites from Utah free-roaming horse feces
DNA was extracted from 150 mg frozen fecal subsamples using the Zymo Quick-DNA Fecal/Soil Microbe Miniprep Kit. DNA yields from both direct extracts and 1:10 lysates were quantified using the Qubit High Sensitivity assay. Library preparation followed a modified protocol from Avramenko et al. (2015), with triplicate first-stage PCRs targeting the ITS2 region using NC1-NC2 primers and Kapa HiFi Hotstart mix. A 1:50 dilution of pooled products was used for second-stage PCR with Platinum II Taq and TruSeq indexing primers. Amplicons were purified using AMPure XP beads (0.8:1 ratio), quantified with PicoGreen, and pooled at equimolar concentrations. Sequencing was performed on an Illumina MiSeq using the v2 500-cycle Nano kit, with samples included in the pre-processing method comparison pool.