Complete genome sequence of Staphylococcus casei strain DSM 15096

We present the first complete genome sequence of the species Staphylococcus casei . Strain DSM 15096 was sequenced with a hybrid approach using Oxford Nanopore Technologies long-read sequencing and Illumina short-read sequencing. The assembled sequences produced a 2 808 898 bp chromosomal molecule containing 2705 predicted genes, plus eight plasmids.


ANNOUNCEMENT
Staphylococcus casei is a coagulase-negative staphylococcal species that was originally isolated from surface-ripened cheese in 1997 and classified as Staphylococcus succinus subsp. casei in 2002 by Place et al. [1]. Later phylogenomic analysis of the Staphylococcaceae family by Madhaiyan et al. determined that S. succinus subsp. casei should be reassigned as a novel species, S. casei [2]. This new nomenclatural status was approved by the International Code of Nomenclature of Prokaryotes [3]. The type strain of the species is S. casei DSM 15096 (SB72 T ) and its original genome sequence was deposited with NCBI in 2018 under Genbank assembly accession GCA_002902445.1 [4]. The genome sequence has however been flagged by NCBI as misassembled and the assembly was excluded from RefSeq. There are currently no other genome sequences publicly available for either S. succinus subsp. casei or S. casei. To address the lack of an available, high-quality genome sequence for this species, we purchased S. casei DSM 15096 as a freeze-dried sample from the German Collection of Microorganisms and cell cultures (https://www.dsmz.de/) and applied a hybrid assembly approach using Oxford Nanopore Technologies long-read sequencing and Illumina short-read sequencing to produce a complete genome sequence.
The strain was revived in Tryptone soy broth (TSB; Oxoid) and streaked onto Tryptone soy agar (Oxoid) for single colonies. A single colony was transferred to 5 ml of TSB and incubated overnight at 37 °C with orbital shaking. The liquid culture (1 ml) was centrifuged, and the cell pellet was resuspended in 200 µl of enzymatic lysis buffer (20 mM Tris-Cl, pH 8.0, 2 mM sodium EDTA, 1.2 % Triton X-100, 20 mg ml −1 lysozyme, 25 µg ml −1 lysostaphin) and incubated at 37 °C for 1 h. DNA was extracted using a QIAGEN DNeasy blood and tissue kit (QIAGEN) following the Gram-positive extraction protocol. Illumina short-read sequencing was conducted by MicrobesNG (https://microbesng.com/). The genome was sequenced using the Illumina HiSeq 2500 platform and 2×250 bp paired-end technology producing 551534 paired-end reads. Nanopore library preparation was performed using the ligation sequencing kit SQK-LSK109 (Oxford Nanopore Technologies) and native barcoding and sequenced on a MinION flow cell (R.9.4.1).
The complete bacterial genome was annotated using the NCBI prokaryotic genome annotation pipeline version 6.5. The chromosome was predicted to encode 2705 genes, 25 rRNAs, 61 tRNAs and 27 pseudogenes. The strain contained eight plasmids that ranged in sizes from 32 242 bp to 2 505 bp. Assembly quality was determined with CheckM v1.1.3 [16] with a completeness of 99.45 %, contamination of 1.38%, an N50 of 2 808 898 bp and G+C content of 33.23 %. Average nucleotide identity (ANI) against the existing misassembled S. casei type strain genome (GCA_002902445.1) showed 99.89 % similarity and was calculated using FastANI v1.1 [17]. ANI between the type strain of the species S. succinus DSM 14614 (AMG-D1 T ) and the chromosome of our S. casei assembly showed 95.58 % similarity. This supports the finding of Madhaiyan et al. who reported 95.6 % ANI between the existing S. casei type strain genome and S. succinus DSM 14614 (AMG-D1 T ).
Funding information C.W. was funded by a BBSRC DTP PhD studentship.

Peer review history
Comments: The methodological approach taken was rigorous and is clearly described, allowing for replication. Underlying data are available with references to repositories made. Results are clearly and succinctly described. The 'niche' for the work completed is clearly identified, and as such there is a clearly described place for this work in the scientific literature where it will have value for the wider community.

Please rate the manuscript for methodological rigour Very good
Please rate the quality of the presentation and structure of the manuscript

Very good
To what extent are the conclusions supported by the data? Strongly support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines? Yes