16S microbiome sequencing chicken
16S rRNA gene library preparation and high-throughput sequencing were performed at Clear Labs (San Carlos, CA, USA) according to the method described by Bolinger et al. (2021). Briefly, DNA extraction was performed on two technical replicates of each sample in a 96-well format, following a modified version of the NucleoSpin Food Kit (Marcherey-Nagel, Dren, Germany) protocol, and optimized at Clear Labs. Following extraction, the amplification of multiple DNA bar-coding markers was conducted using proprietary primers strategically designed to bind the 16S ribosomal RNA region. Products obtained after amplification were purified and prepared for sequencing. Samples analyzed in this study were sequenced in replicates to ensure the quality of the results using an in-house Illumina MiSeq instrument (Illumina, Inc., San Diego, CA, USA) with a 300-cycle MiSeq (version 3) reagent kit. Thereafter, sequence processing and microbial classification were performed using the Clear lab pipeline leveraging an in-house database.