Ampure xp magnetic beads

5.7 μl of retro‐transcription mix (see reference for details) was added to each sample. Retro‐transcription was carried out according to the original Smart‐seq2 protocol and cDNA was then pre‐amplified for 15 cycles (Picelli et al, 2014 (link)). After PCR pre‐amplification, cDNA was purified using Ampure XP magnetic beads according to the manufacturer's instructions, in a ratio of 0.8 to 1 with cDNA, resuspended in 17.5 μl of buffer EB (Qiagen) and stored at −20°C. Quality and concentration of the cDNA generated were assessed using High‐Sensitivity Bioanalyzer kit (Agilent).

Analysis of mtDNA sequence variations was done using GeneChip™ Human Mitochondrial Resequencing Arrays 2.0 (MitoChip v2.0) [44 (link)]. For processing of the arrays, the GeneChip® CustomSeq® Resquencing Array Protocol version 2.1 was used with the following modification: instead of using long-range PCR, mtDNA was amplified using the REPLI-g mitochondrial DNA kit (Qiagen Hilden, Germany), as it has been described for MitoChip experiments before [45 (link)], and purified using Agencourt® AMPure® XP magnetic beads (Qiagen Supplementary Protocol, REPLI-g mitochondrial DNA kit). After purification, 270 ng of mtDNA was fragmented and labeled using the GeneChip® Resequencing Assay Kit (Affymetrix, now part of ThermoFisher Scientific). After hybridization, MitoChip v2.0 arrays were washed and stained in a Fluidics Station 450 before being scanned in Affymetrix GeneChip Scanner 3000 7G. CEL files were acquired by GeneChip® Operating Software 1.4 (GCOS 1.4.0.036) and analyzed with GeneChip® Sequence Analysis Software (GSEQ 4.1). Complete microarray data are available at Gene Expression Omnibus (GEO accession number: GSE211250).

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.