Phusion high fidelity polymerase chain reaction

Based on the user manual, total genomic DNA was extracted from donkey stool samples using the Magnetic Soil and Stool DNA Kit (Tiangen Biochemical Technology, Beijing). DNA was extracted from fecal samples using the cetyltrimethylammonium bromide (CTAB) method, and DNA stress and concentration were detected using 1% agarose gel electrophoresis. Amplification was performed using Phusion High-Fidelity polymerase chain reaction (PCR) Master Mix (New England Biolabs; 341F CCTAYGGGRBGCASCAG, 806R GGACTACNNGGGTATCTAAT). The mixed and purified PCR products were then constructed through terminal repair and street sequencing. NovaSeq 6,000 was used to perform onboard sequencing on PE 250. Clean reads were obtained by excluding low-quality and barcode reads.

The extraction of bacterial DNA from fresh sample was determined by the method of Li [21 (link)]. In brief, Phusion^® High-Fidelity polymerase chain reaction (PCR) Master Mix (New England Biolabs) was used to carry out PCR reactions, following the manufacturer’s instructions. The primers 515 F and 907 R was chosen to amplify the V4–V5 region of 16S rRNA gene. The PCR amplicons were then sequenced by using an Illumina MiSeq PE2500 platform at Novogene Company (Beijing, China). After sequencing, paired reads were merged using FLASH (V 1.2.7) and filtered by QIIME. The UPARSE method was employed to assign operational taxonomic units (OTUs) to the 16S rRNA at a cutoff level of 3% on the Usearch software platform (Version 7.1). Based on OTUs results, the alpha indices were calculated with QIIME (Version 1.7.0) and displayed with R software (Version 2.15.3).