The V4 region of the 16S ribosomal RNA from human stool sample: before Helichrysum italicum infusion consumption
A fresh stool sample was stored in stool collection tubes at 80C until analysis.  DNA was extracted from the frozen faecal samples (1-2 g) using the commercial QIAamp DNA Stool Mini Kit (Qiagen N. V., Venlo, The Netherlands) following the manufacturers instructions and stored at 80 C for further analysis. Amplification of V4 16S rRNA was performed with 515F (modified by Parada et al., 2015) and 806R (modified by Apprill et al., 2015). Primer 806R was elongated with the sequence of P1 adapter and 515F primer was elongated with the sequence of linker, barcode and adapter at their 5 end in order to produce amplicons compatible for sequencing with Ion S5 System (Thermo Fisher Scientific). Ion  530 chip with DNA library and Ion S5 calibration standard was prepared with Ion OneTouch 2 system using the kit Ion 520 & Ion 530 Kit-OT2 and sequencing was performed on Ion S5 System (Thermo Fisher Scientific).