PA4_ITS
To assembly the community taxonomic composition regarding bacteria and fungi present in cystic fibrosis patients, the 16S rRNA V3-V4 and ITS (Internal Transcribed Spacer) regions were amplified from aliquots of 1 l from the whole genomic DNA extracted from the samples. The 16S rRNA V3-V4 regions were amplified by a first PCR reaction employing the universal primers: forward 5'  TCGTCGGCAGCGTCAGATGTGTATAAGA GACAGCCTACGGGNGGCWGCAG  3, and reverse GTCTCGTGGGCTCG GAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC- 3. The reational media of each PCR contained 10 L of GoTaq Colorless Master Mix 2x (Promega, USA), 1l of metagenomic DNA and 0,3 M of forward and reverse primers. The final volume of 20 l was achieved completing the reational volume with ultrapure water.The PCR conditions were characterized by a first denaturation step at 94C/3 minutes, followed by 25 cycles of denaturation at 95C/30 seconds, annealing at 55C/30 seconds, extension at 72C/30 seconds, and a final extension step at 72C/5 minutes.To evaluate the quality and to confirm the amplification, an agarose gel 2% (m/v) electrophoresis coloured with UniSafe Dye 0,003% (v/v) was performed. The average amplicon size obtained for each sample was 500 bp.Regarding the ITS amplification for fungi detection, the forward 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGAATCATCGAATCTTTGAA-3 and reverse 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTC CTCCGCTTATTGATATGC-3 universal primers were used. The PCR reaction components were the same as described above for 16S rRNA V3-V4 regions amplification, although the PCR conditions to amplify ITS regions were slightly different: initially, a denaturation cycle at 95C/5 minutes was performed, followed by 35 cycles of denaturation at 95C/30 seconds, annealing 56C/ 40 seconds, extension at 72C/1 minute, and a final extension step at 72C/5 minutes. All PCR reactions were conducted on VeritiTM Thermal Cycler (Applied Biosystems, USA).The integrity checking of the generated amplicons was performed by an agarose gel electrophoresis 2% (m/v) coloured with UniSafe Dye 0,003% (v/v). The average amplicon size obtained for each sample was 400 bp. The PCR amplicons were subsequently purified using magnetic beads Agencourt AMPure XP (Beckman Coulter, USA) following manufacturers guideline.The indexation step, required to pool the samples and to insert them into the flowcell, was performed through a second PCR reaction for the entire set of generated amplicons. In this reaction, the adapters sequences were inserted in the amplicons extremities. The PCR reaction was conducted with the kit Nextera XT index (Illumina), following the manufacturers standards for PCR. The resulting libraries were purified using magnetic beads provided in the kit Agencourt AMPure XP (Beckman Coulter) to remove fragments minor than 100 bp and residual primers. The libraries were quantified by RT-PCR using the kit KAPA-KK4824 Library Quantification Kit (Illumina) according manufacturer recommendations. Posteriorly, an equimolar pool of samples was generated through a normalization of all samples to the final concentration of 3 nM. The High-throughput DNA sequencing (HTS) was conducted on MiSeq platform (Illumina).