16S sequences from Coffea canephora clone 02 replicate 3
The V3-V4 subregion of 16S was amplified using the primers 341F (5'-CCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACNVGGGTWTCTAAT-3'). The ITS1 region was amplified using the ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3'). The PCR reactions were performed with Phusion High-Fidelity PCR Master Mix (New England Biolabs). Samples with bright main strip between 400-450bp in a 2 % agarose gel were chosen for further experiments. PCR products was mixed in equidensity ratios and resulting library pool was was purified with Qiagen Gel Extraction Kit(Qiagen, Germany). Sequencing libraries were generated usingNEBNext Ultra DNA Library Pre  Kit for Illumina, following manufacturer 's recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. The sequencing was performed on an Illumina platform and 250 bp paired-end reads were generated.