Miseq technique

Total DNA from the microbial community in the ruminal fluid from 24 yaks was extracted according to the instructions of the FastDNA^® Spin Kit for Soil (MP Biomedicals, Solon, OH, United States). The determination of DNA concentration and purity, primer selection for PCR amplification, and PCR amplification procedures were performed according to our previous study (Yi et al., 2022 (link)). The PCR products of the same sample were mixed, subjected to 2% agarose gel to recover the PCR products, and extracted using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). A Quantum Fluorometer (Promega, Madison, WI, USA) was used to detect and quantify the recovered products. The sequencing library was constructed and sequenced using the Illumina Miseq technique (Illumina, San Diego, CA, USA) in a manner consistent with our previous study (Yi et al., 2022 (link)). All raw sequences have been uploaded to the NCBI database with the accession number: PRJNA978645.

Species of the bacterial communities were assayed with a genotypic fingerprinting approach using the Illumina MiSeq technique. Total DNA concentrations that met the requirements of amplification were used for subsequent analysis (Illumina MiSeq high-throughput sequencing, Enterprise Group Personal Biotechnology Co., Ltd., Shanghai, China).

Whole genome sequencing was performed on the 17 strains using Illumina MiSeq technique and associated protocols (Illumina, San Diego, CA) with MiSeq Reagent Kit v2 (500 cycle), Nextera XT DNA Sample Preparation kit, and Nextera XT Index Kit. Draft genome data were assembled using CLC Genomic Workbench de novo and were annotated by NCBI using Prokaryotic Genomes Annotation Pipeline (Klimke et al., 2009 (link)). These draft genomes were deposited in GenBank under the following accession numbers: SHL001 (APIB00000000), SHL002 (APIB00000000), SHL004 (APIB00000000), SHL005 (APIB00000000), SHL006 (APIB00000000), SHL007 (APIB00000000), SHL008 (APIB00000000), SHL009 (APIB00000000), SHL010 (APIB00000000), SHL011 (APIB00000000), SHL012 (APIB00000000), SHL013 (APIB00000000), SHL014 (AWWQ00000000), SHL015 (AWWR00000000), SHL016 (AWWS00000000), SHL017 (AWWT00000000), and SHL018 (AWWU00000000).

Total DNA was isolated from frozen stool samples using MagnaPure LC DNA Isolation Kit III (Bacteria, Fungi) (Roche, Mannheim, Germany) according to manufacturer’s instructions including mechanic and enzymatic lysis [13 (link)]. Hypervariable regions V1-2 were amplified in a target specific PCR using the primers 27F and R357 (27F-AGAGTTTGATCCTGGCTCAG; R357-CTGCTGCCTYCCGTA) and sequenced with the Illumina MiSeq technique (Illumina, Eindhoven, The Netherlands) [13 (link)]. Sequencing was done in cooperation with the Core Facility for Molecular Biology at the Center for Medical Research in Graz.

One pair of specific primers for amplification of the region between pir (replicon protein) and hns (DNA-binding protein) was designed to explore the genetic contexts of mcr-1 on IncX4 plasmids. Mcr-1-harbouring IncX4 plasmids with different genetic contexts were selected and then prepared from the transconjugants using the QIAGEN Plasmid Midi kit (QIAGEN) and were sequenced by Illumina MiSeq technique (Illumina, San Diego, USA). Illumina sequences were de novo assembled using SOAP de novo^{43 (link)}. The gaps between the contigs were closed by PCR and respective amplicons were sequenced. Gene prediction and annotation were performed using the RAST tools^{44 (link)}. To gain insights into the variations of IncX4 plasmids, sequence comparisons of the 30 completely-sequenced IncX4 plasmids (collected until July 18, 2016) were applied BLAST and Easyfig^{45 (link)}. The variable regions of the other IncX4 plasmids without sequencing in this study were further analyzed by PCR-RFLP. The PCR products of the variable regions were purified and then digested with the following restriction enzymes (TaKaRa, Dalian, China): ClaI for Variable Region I, EcoRV for variable Region II, and HincII for Variable Region III. Comparison of PCR-RFLP patterns were performed with BioNumerics software version 2.5 (Applied Maths), and clusters were defined by cutoff of 100% similarity between DNA band patterns.

Total DNA was isolated from frozen stool samples using MagnaPure LC DNA Isolation Kit III (Bacteria, Fungi) (Roche, Mannheim, Germany) according to manufacturer’s instructions including mechanic and enzymatic lysis [22 (link)]. Hypervariable regions V1-V2 were amplified in a target specific PCR using the primers 27F and R357 (27F-AGAGTTTGATCCTGGCTCAG; R357-CTGCTGCCTYCCGTA) and sequenced with the Illumina MiSeq technique (Illumina, Eindhoven, The Netherlands) [22 (link)]. Sequencing was done in cooperation with the Core Facility for Molecular Biology at the Center for Medical Research in Graz.