Metagenomic analysis of colonic fermentation with Nannochloropsis gaditana
DNA from feces was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). The concentration of the DNA extracts was determined with the Invitrogen Qubit 3 Fluorometer using the dsDNA HS (High Sensitivity) Assay (Thermo Fisher Scientific, Madrid, Spain). Microbiota analysis was performed by 16S ribosomal RNA (rRNA) bacterial gene amplification and sequencing, using the Ion Torrent sequencing technology and the reagents from Life Technologies S.A. (a part of ThermoFisher Scientic), as previously described (12). Briefly, for each sample, a starting input of 5ng of DNA was used to run 2 parallel polymerase chain reactions (PCRs), which amplified seven hypervariable regions of the gene (V2, V4, V8 and V3, V6-7, V9, respectively) with two different sets of primers, using the Ion 16S Metagenomics Kit. The PCR conditions followed the manufacturers protocol, and both PCR reactions were set to 25 cycles. The amplicons obtained from both reactions were then quantified, combined for each sample in equal volumes and purified using the Agencourt AMPure XP Reagent beads (Beckman Coulter, Madrid, Spain). Next, barcoded libraries were prepared using the Ion Plus Fragment Library Kit and the Ion Xpress Barcode Adapters. The libraries were then quantified with the Qubit 3 Fluorometer, set to 22pM, and then combined in the same tube. An emulsion PCR was performed using the Ion OneTouch 2 System together with the Ion 520 & 530 Kit  OT2. The template-positive Ion Sphere Particles (template-positive ISPs) were then collected, washed and enriched using the Ion OneTouch ES Instrument OT2, and finally loaded onto an Ion 530 Chip and sequenced using the Ion S5 System.