Draft Reference Genome Sequence of Corynebacterium mastitidis RC, an Ocular Commensal, Isolated from Mouse Conjunctiva.

Here, we report the genome sequence of a protective commensal, Corynebacterium mastitidis RC, isolated from mouse conjunctiva. The C. mastitidis RC genome sequence is 2,153,054 bp in size and 96.95% complete, and we believe that it can contribute to the understanding of the functional immune attributes of the ocular commensal microbiome.

ANNOUNCEMENT

Corynebacterium is a Gram-positive, pleomorphic bacterium that exists frequently as a normal flora inhabiting skin, mucous membrane, and conjunctiva. Several Corynebacterium species have also been identified as either pathogens or opportunistic pathogens causing diseases like diphtheria, endocarditis, and mastitis (1). Genome sequences of two Corynebacterium mastitidis species, namely, one isolated from the milk of a sheep with subclinical mastitis (2) and the other isolated from a mouse with keratitis (3), are currently available in NCBI genome database. Here, we report the nearly complete draft genome sequence of C. mastitidis strain RC, identified as a protective commensal on the ocular surface in our previous study (4). The C. mastitidis strain RC was isolated from the conjunctiva of a wild-type, 8-week-old, female NIH mouse (C57BL/6). The single-colony isolates were identified as C. mastitidis using 16S rRNA gene sequencing.

Single-colony isolates of C. mastitidis were obtained using methods described in our previous study (4). Briefly, mice conjunctiva homogenate was spread on tryptic soy agar (TSA) plates and incubated for 7 days. The isolated C. mastitidis colonies were confirmed as axenic using 16S rRNA gene sequencing. For sequencing, single-colony isolates of C. mastitidis RC were cultured using tryptic soy agar + 5% blood agar plates, at 37°C under aerobic conditions. DNA was extracted using the bacterial DNA prep kit (Zymo Research, Irvine, CA) as per the manufacturer’s instructions. DNA concentration was quantified with Qubit (ThermoFisher, Waltham, MA). Sequencing libraries were prepared with the Nextera XT DNA library prep kit (Illumina, San Diego, CA). Library quality was accessed using TapeStation system (TapeStation Analysis Software A.02.01 SR1; high-sensitivity D5000 ScreenTape kit; Agilent Technologies, Inc.). Paired-end sequencing was done in the Illumina MiSeq platform, with the 600 cycle V3 kit box 1, using one flow cell. FastQC (v0.11.9) (5) was used to analyze the quality of the reads before and after processing. BBTools (v38.87) (6) was used to remove adapters and preprocess the reads. A de novo genome assembly was done using the SPAdes optimizer Unicycler (v0.4.8) (7), comprising of SPAdes (v3.15.2) (8) and Pilon (v1.23) (9), with default parameters for all three available modes. Assembly quality was assessed using CheckM (v1.1.3) (10) and QUAST (v5.0.2) (11), using default parameters. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (v6.0) was used for annotation (12). The complete code, with parameters and custom scripts used for each of the analysis components of the workflow, is provided at the GitHub repository online at https://github.com/NIH-NEI/cmast-genome-assembly.

A total of 3.8 million reads with a 250-bp read length were generated using the paired-end sequencing technique. The assembled C. mastitidis RC genome is 2,153,054 bp long, with 42 contigs at the N50 of 130,636 bp and NG50 of 130,636 bp, 80× coverage, and the GC content of 69.06%, after removing 5 contigs with a size less than 200 bp. Compared with the 585 gene markers in the Corynebacterium lineage, C. mastitidis RC shows a 96.95% completeness. The PGAP annotations identified 2,132 total genes in C. mastitidis RC, comprising of 2,050 protein-coding genes, 52 tRNAs, 3 rRNAs, 2 noncoding RNAs (ncRNAs), and 2 CRISPR arrays.

Data availability.

The raw sequencing data are deposited and available at NCBI SRA under the accession number SRR15665287. The complete project information is available under the NCBI BioProject identifier (ID) PRJNA758739. The sample information is available under the NCBI BioSample ID SAMN21033600. The annotated genome sequence is deposited in NCBI under the accession JAKRKB000000000. The code and the workflow documentation describing the genome assembly are deposited and available at the github repository online at https://github.com/NIH-NEI/cmast-genome-assembly. The permanently linked code release is also published in Zenodo, online at https://doi.org/10.5281/zenodo.6282054.