title: Metataxonomics:honey:Canudo
design: A amount of 250 mg of the honey and papus pooled samples was collect for total metagenomic DNA extraction with the PowerLyzer PowerSoil DNA Isolation Kit (MoBio Laboratories, Qiagen, USA), following the manufacturers instructions. The required DNA concentration must be at least 5ng/l for the downstream analysis. The Paired-End (PE) HTS was performed on a Miseq platform (Illumina, USA) at the facility for nucleic acid sequencing of the Faculdade de Cincias Farmacuticas de Ribeiro Preto - Universidade de So Paulo, Brazil. V3 and V4 regions of 16S rRNA genes were amplified by PCR with specific universal primers developed by the company Illumina (forward=5CGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGC WGCG3; reverse=5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGTATCTAA TCC3) with overhang adapters attached. The produced amplicons were purified with Agencourt AMPure XP beads (Beckman Coulter, USA) to remove exceeding primers and primers dimers. Thus, a second PCR was performed to attach dual indexes and the adapter sequences, using the Nextera XT Index kit  (Illumina ). After this step, another purification procedure was performed with Agencourt AMPure XP beads (Beckman Coulter) to remove exceeding oligos that may occupy space in the flowcell. Finally, the libraries were quantified by real-time PCR (Kapa HiFi Hot Start ready Mix, Kapa Biosystems, USA), normalized and the finished libraries were pooled, according manufacturer instructions of the 16S Metagenomic Sequencing Library Preparation manual. The libraries were transferred and attached to the flow cell through denaturation with sodium hydroxide and loaded employing the MiSeq reagent kit V3 (600 cycles). For internal control of the low diversity libraries and to prevent the run to fail, a minimum of 5% of PhiX control kit v3 (Illumina, USA) for each run were used.