16S rRNA Gene Sequencing of Cecal Microbiome

Almost 0.5–1.0 g of homogenized cecal chyme of each chick was used. Total genomic DNA was extracted using the EZNA^TM Soil DNA kit (D5625-02, Omega Bio-Tek Inc., Norcross, GA, United States) and stored at −20°C. The V4 region of bacterial 16S rRNA was amplified by PCR using the primer pair 515F/806R (Bergmann et al., 2011 (link); Gao et al., 2017 (link)). The amplified products containing main fragments of 400–450 bp were extracted and chosen for further analysis (Caporaso et al., 2011 (link); Gao et al., 2017 (link)). PCR products were purified using the GeneJET Gel Extraction Kit (Thermo Scientific, Waltham, MA, United States). After Qubit quantitative and library detection, the individually barcoded 16S rDNA amplicons from each sample were pooled and paired-end sequenced on the IonS5^TM XL platform at Novogene Bioinformatics Technology Co., Ltd (Beijing, China), and 250-bp paired-end raw reads were generated.