16S V3-V4 amplicon of Cobb500 broiler: Caecal tissues, Unvaccinated, challenged, 10 dpi
Bacterial genomic DNA was extracted from caecal tissues (~100 mg) and contents (~200 mg) using QIAamp Fast DNA Stool Mini kit (QIAGEN, Valencia, CA, USA), following pathogen detection protocols. The 16S amplicon library preparation was done by following the guideline of Illumina 16S Metagenomic Sequencing Library Preparation [21]. The 16S ribosomal RNA (rRNA) genes V3V4 hypervariable regions were amplified using PCR with the adapter overhang primers 341F (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGC CCTACGGGNGGCWGCAG-3) and 805R (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GACTACHVGGGTATCTAATCC-3) for 25 cycles. The PCR products were attached indices and Illumina sequencing adapters using the Nextera XT Index Kit by 8 cycles of second amplification reaction. The final PCR products were purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). The amplicon DNA concentration was measured using the Qubit dsDNA HS and BR Assay Kits (Thermo Fisher Scientific, Waltham, MA, USA). The quality of library was determined using the Agilent Technologies 2100 Bioanalyzer system with DNA-1000 chip. Eighty-eight samples were pooled with equal molality. The 16S amplicon libraries were sequenced in a 301 bp paired-end (301bp x 2) on an Illumina MiSeq platform using V3 chemistry.