Miseq pe300 high throughput sequencing platform

DNA of cecal contents was extracted using the DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA). The extracted DNA was identified by 1% agarose gel electrophoresis and spectrophotometry (260/280 nm optical density ratio). The V3-V4 extender primers of 16S rDNA were 338F (5′-ACTCCTACGGGAGgCAGCAGcag-3′) and 806R (5′-GGACTACNNGGG TATctaat-3′). The V3-V4 region of bacterial 16S rDNA was selected and sequenced by an Illumina (Illumina, Inc., San Diego, CA, USA) Miseq PE300 high-throughput sequencing platform. The off-machine data were handled by QIIME (V1.8.0) software. The sequence information of each processing group is clustered into operational taxonomic units (OUTs) for species classification according to barcodes. Mothur software (version 1.31.2) was used for α diversity analysis. Based on the weighted UniFrac distance, the pheatmap package of R (V3.1.1) software was used for cluster analysis. The UniFrac algorithm was utilized to compare species community differences between samples using the information of systematic evolution, and the beta diversity analysis was performed. Linear discriminant analysis (LDA) and linear discriminant analysis effect size (LEFSE) were used to analyze the microbial community dominance between groups. The original sequence was uploaded to NCBI's Sequence Read Archive database.

Soil microbiome DNA was extracted based on the instructions of the Power Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, United States). DNA mass and concentration was determined using a 1% agarose gel electrophoresis and spectrometer. The V3–V4 region of the bacterial 16srRNA gene was amplified using primers 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806 R (5′-GGACTACNNGGGTATCTAAT-3′), and 8 bp of barcode sequence was added to each of the upstream and downstream 5′ primer ends to distinguish between the different samples. The PCR product was detected by a 1% agarose gel electrophoresis and purified with Agencourt AMPure XP nucleic acid purification kit. Then, the microbial diversity sequencing library was constructed and paired-end sequencing was performed using Illumina MiSeq PE300 high-throughput sequencing platform.