title: 16S rRNA gene seq PRIMA-KOST study
design: DNA was extracted in random order from the faecal slurries using DNeasy PowerLyzer PowerSoil kit (Qiagen, 12855-100) and the  V3-region of the 16S rRNA gene was PCR amplified using 0.2 l Phusion High-Fidelity DNA polymerase (ThermoFisher Scientific, F-553L), 4 l HF-buffer, 0.4 l dNTP (10 mM of each base), 1 M forward primer (PBU; 5-A-adapter-TCAG-barcode-CCTACGGGAGGCAGCAG-3) and 1 M reverse primer (PBR; 5-trP1-adapter-ATTACCGCGGCTGCTGG-3) and 0.05-5 ng faecal DNA in 20 l total reaction volume. Both primers (TAG Copenhagen A/S) were linked to sequencing adaptors and the forward primer additionally contained a unique 10 bp barcode (Ion Xpress Barcode Adapters) for each sample. The PCR program consisted of an initial denaturation for 30s at 98 C, followed by 24 cycles of 98 C for 15 s and 72C for 30 s, and a final extension at 72 C for 5 min. The PCR products were purified by the HighPrep  PCR clean-up system (AC-60500 Magbio) according to the manufacturer's protocol. The resulting DNA concentrations were determined by Qubit HS assay and libraries constructed with mixing equimolar amounts of each PCR product. Partial 16S rRNA gene sequencing was performed on an Ion S5 System (ThermoFisher Scientific) using OneTouch 2 Ion5: 520/530 kit - OT2 400bp and an Ion 520 Chip