Miseq desktop

Genomic DNA was extracted from pure bacterial colonies using Qiagen’s DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA) on a QIAcube automated workstation (Qiagen). DNA concentrations were determined using the Qubit dsDNA HS Assay Kit on a Qubit flex fluorometer (ThermoFisher Scientific, Waltham, MA, USA). DNA libraries were constructed with the Nextera XT DNA Library Preparation Kit (Illumina^®, San Diego, CA, USA) using 0.2 ng/µL of genomic DNA. Resultant libraries were sequenced on an Illumina MiSeq desktop sequencer using v2 sequencing chemistry with 2 × 250 bp pair-end reads per the manufacturer’s protocol. The quality control of raw sequences was performed by FastQC v0.11.9, and the de novo assembly was done using SPAdes v3.14.1. The final contigs of the isolated strain (designated strain 4524) were subjected to BLASTn to identify the reference genome, which shows the highest identity with the contigs. The genome of the 4524 strain was reordered using progressive Mauve with the A. equuli subsp. equuli ATCC 19392 genome as the reference genome [18 (link)]. The FASTA file of the 4524 genomes was used for genome annotation and further analysis.

DNA isolated from BS and RS samples was used for metagenomic analysis. These samples were processed and sequenced with Shotgun using Illumina ^® MiSeq desktop using the 2 × 250 bp paired-end reagent V2 Kits (Illumina ^®, United States) technology with a standard quantification pattern. Bioinformatic analysis and quality control were performed using the Fast QC tool (Andrews, 2017 ). Q-score was used to predict the probability of an error in base-calling. Over 75% of bases > Q30 averaged across the entire run was considered acceptable. Raw sequence reads underwent quality trimming using Trimmomatic to remove adaptor contaminants and low-quality reads (Li et al., 2010 (link)).

DNA amplification was performed using 2x PCR Mastermix (Thermo Scientific, Waltham, USA) according to the protocol of the manufacturer. Amplification products were purified using QIAquick PCR Purification Kit (Qiagen, Germany) and sequencing of purified PCR products was carried out by Macrogen (Amsterdam, The Netherlands). Sequences were analysed with the CLC Main Workbench 7.7.2 (CLCbio, Qiagen, Denmark).
Genomic DNA was prepared using the MasterPure DNA Purification Kit (Epicentre, Illumina Inc., Madison, Wisconsin) according to the manufacturer’s instructions and the samples were prepared for whole genome sequencing as according to Nextera® XT DNA Library Preparation Guide (Rev. D) (Illumina Inc., Madison, Wisconsin). Sequencing was performed using a MiSeq™ desktop sequencer, according to the manufacturer’s instructions (Illumina Inc., Madison, Wisconsin). Sequences were analysed with the CLC Genomic Workbench 11.0.0 (CLCbio, Qiagen, Denmark).