Hiseq 2500 pe250 system

Next-generation sequencing was performed with an Illumina HiSeq 2500 PE250 system by Gene Denovo Biotechnology Co., Ltd. (Guangzhou, China) to investigate the bacterial community. The raw sequence data were processed and analysed with the QIIME software package [19 (link)]. Then, sequences with a threshold of 97% similarity were clustered into operational taxonomic units (OTUs) using the UPARSE pipeline (version 9.2.64) [20 (link)]. These OTUs were used for diversity (Shannon and Simpson indices), richness (ACE and Chao indices), and rarefaction curve analyses using MOTHUR [21 (link)]. Taxonomic assignments of OTUs that reached the 97% similarity level were made using QIIME by comparison with the SILVA databases (http://www. arb-silva.de, accessed on 3 July 2022) [22 (link)]. Venn analysis of OTUs between groups was performed in R project VennDiagram package (version 1.6.16) [23 (link)]. Non-metric multidimensional scaling (NMDS) plots of sequence-read abundances were generated with the Vegan package in R software. A test for statistically significant group differences was performed using the non-parametric ANOSIM procedure.

The metagenomic DNA of 21 rumen fluid samples was extracted by the cetyltrimethylammonium bromide method. DNA was dissolved in the 200 µL of elution buffer and then preserved at −20 °C. DNA integrity and concentration were determined by 1.5% agarose gel electrophoresis and NanoPhotometer^® spectrophotometer (Implen, Westlake Village, CA, USA), respectively. For rumen bacterial analysis, universal primer pairs (515F-806R) with barcodes were used to PCR amplify the V4 region of the 16S rRNA gene (Li et al., 2016 (link)). PCR amplification was done by using Phusion^® High-Fidelity PCR Master Mix with GC Buffer from New England BioLabs. The amplified products were detected by 2% agarose gel electrophoresis and purified by QIAquick PCR Purification Kit to build libraries. The libraries were built according to the manufacturer’s protocol using TruSeq^® DNA PCR-Free sample preparation kit. The libraries were then quantified by using Qubit dsDNA High Sensitivity Assay kit by Invitrogen. The Illumina HiSeq2500 PE250 system was used for paired-end sequencing using the standard protocol.