The nr ITS2 region was amplified from all DNA by means of a semi-nested PCR approach. In the first PCR, the nr ITS (ITS1-5.8S-ITS2) was amplified with universal primers ITS1F-ITS4 (White et al., 1990 (link)). For the second PCR, ITS3 and ITS4 (White et al., 1990 (link)) tagged primers were used to amplify the ITS2 region of each DNA sample (Voyron et al., 2017 (link)).
PCR products were pooled and purified using Wizard SV Gel and PCR Clean-Up System (Promega) following the manufacturer’s instructions. After quantification with Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA, United States), the purified PCR products were mixed in equimolar amounts to prepare sequencing libraries. The libraries were paired-end sequenced using the Illumina MiSeq technology (2 bp × 250 bp) by IGA Technology Services S.r.l. Unipersonale (Udine, Italy).
PCR products were pooled and purified using Wizard SV Gel and PCR Clean-Up System (Promega) following the manufacturer’s instructions. After quantification with Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA, United States), the purified PCR products were mixed in equimolar amounts to prepare sequencing libraries. The libraries were paired-end sequenced using the Illumina MiSeq technology (2 bp × 250 bp) by IGA Technology Services S.r.l. Unipersonale (Udine, Italy).
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