title: metagenomics of uvilla ripe 4
design: For sequencing an Illumina Novaseq platform (paired-end 150 bp reads, Illumina, USA) was used. The universal fungal primers ITS 86F (5-GTGAATCATCGAATCTTTGAA-3) and ITS 4 (5-TCCTCCGCTTATTGATATGC-3) for the amplification of ITS2 region were used. The KAPA HiFi HotStart ReadyMix (Sigma Aldrich, USA) was used in all polymerase chain reaction reactions. To eliminate free primers and primer dimers from the amplicons, magnetic beads were used in a washing step. Up to 96 libraries were pooled using all the Nextera XT indices in prep-aration for sequencing. The purification, sequencing, and library preparation procedures were carried out as described. FASTq files underwent a quality and filtering process to guarantee taxonomic classification. The QIIME v.2 (Quantitative Insights into Microbial Ecology) pipeline, version 2023.5, was used to analyze marker gene-based micro-biome sequencing data. After denoising the sequences with the denoise wrapper, the ITS2 region was extracted. DADA2 was used for denoising and clustering . DADA2 generates higher-resolution tables of amplicon sequence variables (ASVs) [20]. USEARCH 6.1 was used to find and filter chimera sequences. The ASVs were also compared to a reference database of sequences whose taxonomic composition was established. Removal was done from sequences related to eukaryotes, mitochondria, and chloroplasts. The database used is the UNITE Fungal ITS Database 9.0, based on a set of FASTA files from UNITE Community was used.