250 v2 reagent kit

To assess for GINs across goat samples, 4.8 ng from each DNA extraction was pooled and mixed by vortex (n = 47). In addition, eight individuals were selected as case studies for cross-comparison with PCR findings. Amplicon sequencing was performed according to published protocols [47 ] available at https://nemabiome.ca with modifications. Twenty nanograms of template DNA was added per 30 μl PCR reaction (Q5^® High-Fidelity DNA Polymerase, NEB, cat. M0491L) with a mix of ITS-2 primers containing 0–3 ‘N’ bases and an Illumina adapter (10 μM stock) used at recommended concentrations. Cycling included 35 cycles as described with primer details in Additional file 2: Table S2 with a 98 °C/2 min initial denaturation and 72 °C/2 min final extension. PCR products were assessed by 1.2% gel electrophoresis as described above and purified (Wizard^® SV Gel and PCR Clean-Up System, Promega, cat. A9281). Library prep and sequencing were performed via NGSelect Amplicon 2nd PCR service (Eurofins Genomics) on the MiSeq Illumina platform with the 2 × 250 v2 Reagent Kit (Illumina) with resulting metrics from trimmed reads (by Cutadapt [59 ]) shown in (Additional file 3: Table S3). An average read depth of 242,720 high-quality reads (range 201,800–299,860) was achieved.

Bulk DNA lysates were prepared from larval populations, and the ITS2 rDNA fragment was PCR-amplified and sequenced by deep amplicon sequencing as previously described and validated for the nemabiome metabarcoding assay [13 (link)]. Sequencing was completed on the Illumina MiSeq with the 2× 250 v2 Reagent Kit. An average read depth of ~19,000 was obtained for each sample. Samples were removed if they did not have at least 2000 reads, as this was taken to indicate suboptimal PCR amplification.