Cecal Microbiome DNA Extraction and Sequencing

According to the procedure suggested by manufacturer, the cecal microbial community DNA was extracted using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Bacterial universal primers, 338F (50-ACTCCTACGGGAGGCAGCAG-30) and 806R (50-GGACTACHVGGGTWTCTAAT-30) were used to amplify along with V3/V4 hypervariable regions of 16s rDNA by Polymerase Chain Reaction (PCR) [32 (link)]. The annealing temperature was 55 °C during the 30 PCR cycles. The fungal ITS gene amplification was conducted using primers ITS5F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′). Visualized the PCR products by 1.5% agarose gel electrophoresis and then quantified it by PicoGreen dsDNA Quantitation Reagent (Invitrogen, USA). To obtain purified PCR products, the AxyPrep DNA Purification kit was involved in this part (Axygen Biosciences, USA). The library generated by the purified PCR products were Paired end sequenced (Paired_End) based on the Illumina NovaSeq sequencing platform (Illumina NovaSeq PE250, United States). We spliced and filtered the original data to filter out contaminated data, such as chimera sequences, nucleotide mismatch, and ambiguous character reads, to obtain accurate and reliable adequate data.