Nebnext fast dna library prep set kit

The primer pair BactB-F and BactB-R [47 (link)] and EliZyme HS FAST MIX Red (Elisabeth Pharmacon, Czech Republic) master mix were used to amplify the V4-V5 hypervariable regions of 16 S rRNA genes with the DNA extracted using two methods used as a template. Thermal cycling conditions included an initial denaturation step for 5 min at 95 °C, followed by 25 cycles of 30 s at 95 °C, 30 s at 57 °C and 30 s at 72 °C, ending with a final elongation step for 5 min at 72 °C. The quality and size of obtained PCR amplicons were evaluated on 1.5% agarose gel electrophoresis and purified using Monarch PCR & DNA Cleanup Kit (New England BioLabs, USA). Obtained amplicons were subsequently used for library preparation using the NEBNext Fast DNA Library Prep Set kit (New England Biolabs, USA) according to Milani et al. [48 (link)]. The sequencing was then performed on an Ion Torrent platform (Thermo Fisher Scientific, USA) as it was described by Mekadim et al. [49 (link)].

Obtained PCR products were used to prepare libraries for diversity analyses by next generation sequencing (NGS) approach on Personal Genome Machine (Life Technologies, Carlsbad, CA, USA) according to Milani et al. [71 (link)]. 200 ng of DNA from each sample was used to prepare sequencing libraries by NEBNext® Fast DNA Library Prep Set kit (New England Biolabs, Ipswich, MA, USA) according to manufacturer’s protocol. The Ion Xpress Barcode adapters (Thermo Fisher Scientific, Waltham, MA, USA) were used to label each sample. The adaptor ligated libraries were purified and simultaneously size-selected using AMPure XP bead sizing (Beckman Coulter, Brea, CA, USA). The barcoded libraries were pooled in equimolar amount (about 26 pM). The pool of libraries was used to prepare sequencing template by emulsion PCR on Ion Sphere Particles (ISPs) using Ion PGMTM Hi-QTM View OT2 400 Kit (Thermo Fisher Scientific) in Ion OneTouchTM 2 instrument. The enrichment of the template positive ISPs were performed on Ion OneTouchTM ES instrument. The enriched template positive ISPs were then loaded in Ion 316TM Chip v2 BC (Thermo Fisher Scientific). The sequencing was then performed on an Ion Torrent PGM sequencer (Thermo Fisher Scientific, Waltham, MA, USA) using Ion PGMTM Hi-QTM View Sequencing solutions kit (Thermo Fisher Scientific).

Total genomic DNA was extracted from 300 mg of fecal samples (FS; from three FS per one animal collected independently in intervals as shown in Table 1; 100 mg per each FS) using the QIAamp® PowerFecal® Pro DNA Kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted DNA was then used as a template for the preparation of amplicons from the V4 region of the 16S rRNA gene [14 (link)]. Libraries were prepared from the purified amplicons using the NEBNext Fast DNA Library Prep Set kit (New England Biolabs, USA), according to Milani et al. [15 ]. The sequencing was then performed on the Ion Torrent platform (Termo Fisher Scientifc, USA) as it was described previously by Mekadim et al. [16 ].