Gel extraction and purification kit

Previously published primers and conditions (Avramenko et al. 2015 (link)) were used to amplify the rDNA ITS-2 region. PCR products were purified with AMPure XP magnetic beads according to the manufacturer’s guidelines, followed by the second round of PCR amplification to add unique barcode combinations to each sample using the previously described method (Rehman et al. 2020 (link)). Finally, the samples were pooled (10 µl PCR product from each sample) and purified using a Qiagen gel extraction and purification kit, followed by further purification through AMPure XP magnetic beads. Then, 20 μl of the pooled sample was submitted to Edinburgh Genomics for Illumina MiSeq, using a 500-cycle paired-end reagent kit (MiSeq Reagent Kits v2, MS-103–2003) at a concentration of 15 nM with the addition of 15% PhiX Control v3 (Illumina, FC-11–2003). Each resequencing step followed Illumina’s standard protocol.
The numbers of L₃ recovered varied greatly between farms, and the DNA amount could not be equalised between samples; hence, the results are focused on describing the GIN species present on individual farms, rather than direct proportional comparisons.