Total bacterial DNA was extracted from the intestinal chyme and mucosa using the EZNATM Soil DNA kit (D5625-02, Omega Bio- Tek Inc., Norcross, GA, USA) according to the instructions of the manufacturer. The V3-V4 hypervariable regions of the bacterial 16S rDNA were amplified by a two-step PCR method using primers 338F (5′-ACTCCTRCGGGAGGCAGCAG-3′) and 806R (5′-GGACTACCVGGGTATCTAAT-3′) with unique 8-bp barcodes to facilitate multiplexing, and sequencing was carried out with an Illumina sequencing platform using Miseq PE300 (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).
The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by fastp version 0.20.0 (Chen et al., 2018 (link)), and merged by FLASH version 1.2.7 (Magoc and Salzberg, 2011 (link)). Operational taxonomic units (OTUs) with 97% similarity cutoff (Edgar, 2013 (link)) were clustered using UPARSE version 7.1 [3], and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 (Wang et al., 2007 (link)) against the 16S rRNA database using a confidence threshold of 70% (PRJNA889391).
Free full text: Click here