Genome Sequences of Metabacillus sp. Strain B2-18, Isolated from Human Skin, and Metabacillus endolithicus KCTC 33579T 

ABSTRACT Metabacillus sp. strain B2-18 was isolated from human skin. Here, we present the whole-genome sequences of this strain and Metabacillus endolithicus KCTC 33579T. The genomes of these strains contain 14 and 15 rRNA gene operons, respectively.

I n this study, we report the whole-genome sequences of Metabacillus sp. strain B2-18 and Metabacillus endolithicus KCTC 33579 T (1) to confirm the phylogenetic relationship between the two strains. A swab sample from the forehead skin of a female college student was directly streaked on minimal salts basal (MSB) agar (2) containing 0.5% yeast extract (MSBY medium). The agar medium was anaerobically incubated for 1 week at 28°C for growth. The anaerobic conditions were maintained in a tightly sealed jar with an Anaerobag (Chongqing Pang Tong, China). One isolate, B2-18, which was capable of microaerobic growth, was further purified by streaking on MSBY medium and aerobic incubation at 28°C for 3 days. This strain was deposited as KCTC 43380 (JCM 35039) and was assigned NCBI taxonomy identification number NCBI:txid2897333. Ethical approval for subject sampling was granted by the institutional review board of Changwon National University. Sanger sequencing of the 16S rRNA gene amplicon with primers 27F (59-AGAGTTTGATCCTGGCTCAG-39) and 1492R (59-GGTTACCTTGTTACGACTT-39) (3) yielded unidentifiable overlapping peaks with partially recognizable nucleotide sequences at the 59 and 39 ends. The amplification conditions were the same as those described previously (4). The resulting partial nucleotide sequences were used for homology analysis with the NCBI database and enabled the identification of B2-18 as belonging to the genus Metabacillus. The nucleotide sequences of the 16S rRNA gene of B2-18 from genome sequencing were deposited in GenBank (Table 1).
For DNA extraction, cells were cultured in a flask of MSBY broth for 48 h at 28°C, with shaking at 140 rpm. Total genomic DNA was purified using the phenol extraction method (5). Genomic DNA was sequenced with Illumina and Oxford Nanopore Technologies MinION platforms. Illumina sequencing was performed on a NovaSeq 6000 sequencer at DNALink Co. (Seoul, South Korea) as described previously (6). The whole-genome sequencing was performed using a TruSeq DNA PCR-free 550bp library preparation kit (Illumina) and demultiplexing with bcl2fastaq2 v2.20 on the Illumina NovaSeq 6000 sequencer. The quality of the raw sequencing data was checked using FastQC (ASCII Q score offset, 33) (https://www.bioinformatics .babraham.ac.uk/projects/fastqc) (7). The read length was 2 Â 151 bp (paired-end reads), with an insert size of approximately 550 bp, and the mean Phred quality score was 35.47. For Nanopore sequencing, libraries were prepared with purified raw DNA and the SQK-LSK109 kit (Nanopore) and multiplexed using the EXP-NBD104 barcoding kit according to the protocol. Sequencing was performed on a MinION sequencer using a FLO-MIN106 flow cell (R9.4.1) and MinKNOW v20.10.3 software with default settings. Reads were base called and demultiplexed in fast mode with a minimum Q score of 8 and then were further base called and barcode trimmed in super-accurate mode.
For comparison, M. endolithicus KCTC 33579 T obtained from the Korean Collection for Type Cultures was cultivated, and genomic DNA was purified under the same conditions as described for B2-18. The genomic DNA of KCTC 33579 T was sequenced with the Nanopore platform. The reads of strains B2-18 and KCTC 33579 T were assembled de novo using Unicycler with SPAdes v0.4.9b and Unicycler with miniasm v0.3, respectively, with default settings (8). The quality of the assembled genome sequences was evaluated using CheckM v1.1.3 (9). Default parameters were used for all software unless otherwise noted. Gene predictions and annotations were provided by NCBI using the best-placed reference protein set with GeneMarkS-21 of the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v5.3 and v6.1 for B2-18 and KCTC 33579 T , respectively (10). Table 1 presents the general features of the genomes of B2-18 and KCTC 33579 T . The assembly quality assurance report provided by NCBI showed that the best-matching type strain assembly for B2-18 is M. endolithicus KCTC 33579 T (GenBank assembly accession number GCA_023078335), with an ANI value of 95.40% and query coverage of 75.43%.
Data availability. GenBank accession numbers for raw sequencing data and genome assemblies are listed in Table 1.

ACKNOWLEDGMENT
This research was supported by Changwon National University in 2021 and 2022.