title: Effect of Bacillus PB6 supplementation on intestinal microbiome of broilers
design: The V3-V4 hypervariable region of the 16s rRNA gene was amplified using the gene-specific primers 341F 5-CCTAYGGGRBGCASCAG-3 and 806R 5-GGACTACNNGGGTATCTAAT-3 and barcodes (Yu et al., 2005). All PCR mixtures contained 15 L of Phusion High-Fidelity PCR Master Mix (New England Biolabs, Massachusetts, USA), 0.2 M of each primer and 10 ng target DNA. Cycling conditions consisted of a first denaturation step at 98 for 1 min, followed by 30 cycles at 98 (10s), 50 (30s) and 72 (30s) and a final 5min extension at 72. PCR product was subjected to 2% agarose gel electrophoresis. Mixed PCR products were purified using Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany).  Following manufacturers recommendations, sequencing libraries were generated with NEBNext Ultra IIDNA Library Prep Kit (Cat No. E7645, New England Biolabs). The library quality was evaluated on the Qubit@ 2.0 Fluorometer (Thermo Scientific, Massachusetts, USA) and Agilent Bioanalyzer 2100 system (Agilent, California, USA). Finally, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated at Novogene (UK) Company Limited.