COI deep amplicon sequencing of strongyle species communities obtained from treated and untreated equines from different countries
Fecal samples obtained from treated and never treated equines were collected rectally or directly picked up from the ground after defacing. Larval cultures were performed and DNA isolated using the NucleoSpin Soil Mini kit (Macherey-Nagel, Dren, Germany) kit. Mock community was created using equal amounts of plasmid DNA from 27 different strongyle OTUs, mixed to obtain a specific plasmid copy concentration, and used as templates for PCR. The first PCR was conducted using primers COI_Nema_Fw and COI_Nema_Rv (Duscher et al., 2015) which included ILLUMINA adapters. In a subsequent PCR, indices were added to the products using P7 and P5 primers. PCRs were performed using KAPA HiFi Hot Start ReadyMix. The final PCR products were purified using CleanNGS magnetic beads (ratio 1:1.25), and DNA concentrations were measured using a Qubit 4 fluorometer, with quality assessed using TapeStation. Field samples and mock community sample were sequenced with Illumina MiSeq using V3 600 sequencing kit (2x 300 cycles).
SUB15151266
KU4