Ion sequencing 400 kit

The strains MAFF 212134 and MAFF 212141 (http://www.gene.affrc.go.jp/databases-micro_search_en.php) were picked up as the representative biovar 6 strains for genome sequencing. Their genomes were sequenced and assembled by using the same protocols used in our previous study^{5 (link)}, except that some reagents were different as follows; the Ion PGM Hi-Q View OT2 Kit (Thermo Fisher Scientific Inc.), the Ion PGM Hi-Q View Sequencing Kit (Thermo Fisher Scientific Inc.), and a 318 Chip Kit v2 (Thermo Fisher Scientific Inc.) were used in this study instead of the Ion PGM Template OT2 400 Kit, the Ion Sequencing 400 Kit, and a 318 Chip Kit, respectively. The assembled contigs of MAFF 212134 and MAFF 212141 were annotated using the NCBI PGAP (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/), and registered in the nucleotide sequence databases (DDBJ/EMBL/GenBank).

The QIAamp DNA Stool Mini Kit (Qiagen) was used to extract bacterial DNA present in the feces of the salt-fed rats. The V1-V2 region of 16S rDNA genes were amplified by polymerase chain reaction using a range of universal primers, viz. 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 338R (5′-TGCTGCCTCCCGTAGGAGT-3′), with barcode sequences for multiplexing reads of each sample. DNA library was constructed using Ion Xpress Plus Fragment Library Kit (Thermo Scientific, Wilmington, DE, USA) according to the manufacturer’s instructions, and the final library was quantified using Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA) with high-sensitivity DNA chips. Sequencing of the libraries was performed on a 318D sequencing chip using an Ion Sequencing 400 kit and Ion Torrent PGM system (Thermo Scientific) according to the supplier’s instructions. Sequences were processed and analyzed using Quantitative Insights Into Microbial Ecology (QIIME2, version 2020.08) [16 (link)]. Amplicon sequence variants (ASVs) were classified within QIIME2 using the SILVA v138 database [17 (link)]. Alpha diversity, such as the Chao1 index and Shannon’s diversity, was measured using the qiime diversity alpha command. Bar plot figures for alpha diversity were created using GraphPad Prism v8, with between group statistical differences determined using the Kruskal-Wallis test.