ITS metabarcoding of citrus lesion
Metagenomic DNA extracted from samples selected for metabarcoding were subjected to conventional PCR amplification of the rDNA barcode (partial 18S, ITS1, 5.8S, ITS2 and partial 28S) with primers ITS4 and ITS5. Reaction mixes were based on the MyFi Mix (Bioline, United Kingdom) containing 0.2 M of each primer, 2 l of template DNA and MGW to reach a final volume of 25 l. Reactions were performed in a C1000 (Bio-Rad) using the following thermocycler conditions: 5 min 95C, 40x (30 s 94C, 30 s 52C, 1 min 40 s 72C), 10 min72C. Reactions producing visible PCR products of approximately 500 to 1000 bp after 1.5% RESULT LE General Purpose (BIOzymTC, the Netherlands) agarose gel-electrophoresis and SYBR-safe (Life Technologies, CA, USA) staining were used for Illumina short-read sequencing. The Illumina DNA Prep, (M) Tagmentation kit (Illumina, CA, USA) was used according to the manufacturers specifications to generate 150 paired-end Illumina-ready libraries. Illumina Unique Dual Indexes were used for indexing. A total of five amplification cycles were performed during the library generation step. Library yields were quality assessed by fluorescence on a Qubit 4.0 fluorometer (Thermo Fisher Scientific, MA, USA ), and correct insert sizes were verified using D1000 ScreenTape on a TapeStation 4200 system (Agilent Technologies, CA, USA). Amplicon libraries were equimolarly pooled over four individual sequencing runs. Sequencing was performed using 300-cycle P1 cartridge and flow cell combinations on a NextSeq 2000 system (Illumina, CA, USA). Datasets yielded at least 200 MBase per sample.