Microbiome of Homo sapiens: Saliva
After PCR amplification, we used Qubit dsDNA HS assay kit and Qubit fluorometer (Life Technologies) to quantified amplicons concentration. The samples were diluted to 0.2ng/ l and the Nextera XT DNA Sample Preparation Kit (Illumina) was used according to the manufacturer s instructions to generated Paired-end DNA libraries. In brief, the PCR products were fragmented and tagged with adapter and the library purified by using AMPure XP beads (Beckman Coulter).  Normalization procedure was performed to ensure more equal library representation during sequencing. Samples were pooling and heated at 96 C for 2min and PhiX (12.5pM) library control was added to provide quality control checks. Finally, the amplicons library is loaded on the Illumina MiSeq chip. A sequencing run of 2 250 bp MiSeq paired-end was performed. The data obtained was base called and reads with the same barcode were collected and assigned to a sample on the instrument. After procedure the software generates analysis output in the FASTQ file format.