Ion 16s metagenomics kit

For 16S rRNA amplicon sequencing, similar methods were followed to those previously reported (20 (link)). Twelve microliters of DNA extracted from each sample was used to perform the libraries, and the Ion GeneStudio™ S5 System (Life Technologies, Carlsbad, CA, USA) was used. The 16S hypervariable regions were amplified with two sets of primers, v2-4-8 and v3-6,7-9, and libraries were then constructed by using the Ion 16S™ Metagenomics Kit (Life Technologies) and the Ion Xpress™ Plus Fragment Library Kit (Life Technologies). Libraries containing equal amounts of PCR products pooled with a barcode were prepared by using the Ion Xpress™ Barcode Adapters Kit (Life Technologies). Then, these libraries were quantified by using the Ion Universal Library Quantitation Kit (Life Technologies). Next, 10 pM of each library was pooled and loaded on an Ion OneTouch™ 2 System (Life Technologies), which automatically performs template preparation and enrichment. Template-positive ion sphere particles were enriched with Dynabeads™ MyOne™ Streptavidin C1 magnetic beads (Invitrogen, Carlsbad, CA, USA) by using an Ion One Touch ES instrument. Finally, an Ion 520™ chip (Life Technologies) was loaded with the samples on an Ion GeneStudio™ S5 System sequencer using the Ion 520™ and Ion 530™ Loading Reagents supplied in the OT2-Kit (Life Technologies).

DNA was extracted from frozen fecal samples using the DNA stool Mini kit (Qiagen), following the manufacturer’s instructions. DNA concentrations were measured using QuBit 2.0 Fluorometer kit (Life Technologies). Bacterial 16S ribosomal RNA (rRNA) genes contain nine “hypervariable regions” (named from V1 to V9) that demonstrate considerable sequence diversity among different bacteria. These regions of bacterial 16S rRNA genes were amplified by PCR using two sets of primers V2-4-8 and V3-6,7–9 available in the Ion 16S Metagenomics kit (Life Technologies). The PCR products were purified using SPRI method (Solid Phase Reversible Immobilization) (Agencourt AMPURE XP). Each amplicon was then assessed for fragment size distribution and DNA concentration using a Bioanalyser 2100 (Agilent Technologies, USA). Library preparation followed the Ion Plus Fragment Library (Life Technologies). Products were first end-repaired and purified using SPRI method (Agencourt AMPURE XP). Then, library was bar-coded using the Ion Xpress Barcode Adapter 1–16 kit (Life Technologies) and sample was again purified using SPRI method. Finally, emulsion PCR and enrichment steps were carried out using the Ion PGM (Life Technologies). Sequencing was undertaken using 316 chips and Ion PGMSequencing 400 on the Ion Torrent Personal Genome Machine (PGM).

DNA from three representative faecal samples from each group from the 28th day of the experimental design was extracted using a FastDNA® SPIN kit (MP Biomedicals, Solon, Ohio, USA) according to the manufacturer's instructions. An Ion 16S™ Metagenomics kit (Life Technologies, Madrid, Spain) was used for the metagenomic study carried out by Bioarray Genetic Diagnosis (Bioarray, Alicante, Spain).
After confirming that all DNA samples had good levels of concentration, purity, and integrity, a massive sequentiation was carried out with the platforms QIIME v1.8.0 and USEARCH v.7.0.1090. In order to assign the taxonomy, the different sequences with 97% similarity were assembled into operational taxonomic units (OTUs) using the data base GreenGenes v13_8 with the UCLUST method.