ITS2 DNA metabarcoding of strongyle parasites from Utah free-roaming horse feces
Eggs were concentrated from fecal samples using a modified double centrifugation method with Sheathers sugar solution, and collected from the top layer using sterile cotton swabs. Swabs were stored at 25C prior to DNA extraction with the Zymo Quick-DNA Fecal/Soil Microbe Miniprep Kit. To maximize recovery, swab tubes were rinsed with BashingBead Buffer before extraction. DNA yields from extracts and 1:10 lysates were quantified using the Qubit High Sensitivity assay. Library preparation followed a protocol adapted 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.