Metabarcoding of water: used for coffee depulping
Total DNA extraction was performed using the phenol:chloroform protocol. Total DNA was then quantified with a Nanodrop 2000 instrument. Twenty nanograms of the total extracted DNA, containing complementary adaptors for the Illumina platform, was used to amplify a 16S rRNA gene fragment using primers for hypervariable region V4 (515F and 806R), 18S rRNA gene fragment and  ITS rDNA gene (ITS1 and ITS2). Bar-coded amplicons were generated by PCR. Samples were then sequenced in the MiSeq platform using the 500 V2 kit, following standard Illumina protocols. After sequencing, chimeric sequences detection, removal of noises from pre-cluster and taxonomic attribution was performed using standard parameters of QIIME software package, version 1.9.0. Applying the UCLUST method, sequences presenting identity above 97% were considered the same operational taxonomic units (OTUs) according to the SILVA and UNITE databases.