16S rDNA seg-metagenome-broiler
For microbiota analysis, cecal digesta samples were sent on dry ice to Life and Brain GmbH (Bonn, Germany). Extraction of total genomic DNA from 120 mg-aliquots was performed using ZR BashingBead Lysis Tubes (0.1 and 0.5 mm, Zymo Research, Freiburg, Germany) and the Chemagic DNA Stool Kit (Perkin Elmer, Rodgau, Germany) according to the manufacturer's protocol. Amplicons were produced in a two-step PCR reaction. Amplification of the V3/V4 region of the 16S rRNA gene was carried out in the first step using 2x KAPA HiFi HotStart ReadyMix (Roche, Mannheim, Germany) and the primers Bakt_341F (5'-CCTACGGGNGGCWGCAG-3') and Bakt_805R (5'-GACTACHVGGGTATCT AATCC-3') with the following PCR protocol: 3 min denaturation at 95C and 25 cycles of denaturation (30 s at 95C), annealing (30 s at 55C), extension (30 s at 72C), and final extension at 72C for 5 min. The samples were spot-checked on an Agilent TapeStation 4200 using D1000 and purified with AMPure XP beads (Beckman Coulter, Krefeld, Germany). In the second PCR, dual indices and Illumina sequencing adapters were added to the amplicon ends in preparation for sequencing using the Nextera XT v2 Index Kit (Illumina, San Diego, CA, USA). The reaction solution for the second PCR consisted of 25 L of 2x KAPA HiFi HotStart ReadyMix (Roche, Mannheim, Germany), 5 L of the corresponding Nextera XT index primer, 10 L of PCR grade water and 5 L of purified product from the first PCR. The samples were subjected to the following PCR protocol: 3 min denaturation at 95C and 25 cycles of denaturation (30 s at 95C), annealing (30 s at 55C), extension (30 s at 72C), and final extension at 72C for 5 min. The samples were spot-checked and purified like before. The concentrations of the samples were normalized to 4 nM and a pooled sample was prepared. The pooled sample was quantified using the Qubit dsDNA HS Assay Kit from Thermo Fisher Scientific (Waltham, MA, USA) and fragment size examined on a D1000 ScreenTape. An Illumina MiSeq system was used to perform the sequencing with MiSeq Reagent Kit v3 for 2 x 300 cycles. Clustering was performed at 8 pM with a 30% spike-in of PhiX. Afterwards the MiSeq system was used for the demultiplexing. QIIME 2 version 2022.8 was used to process the 16S sequencing data (Bolyen et al., 2019). DADA2 was used for sequencing quality control, including filtering for PhiX reads and chimeric sequences, and denoising (Callahan et al., 2016). Afterwards, a pretrained SILVA classifier (silva-138-nr99-16S-V3-V4-classifier) was used to identify amplicon sequencing variants (ASV) with >99% similarity. The data was analyzed with the default settings of the MicrobiomeAnalyst platform for -diversity, -diversity, and taxonomic analysis (Dhariwal et al., 2017; Chong et al., 2020).