Draft Genome Sequences of Blastococcus sp. Clones TML/M2B and TML/C7B, with Different Motilities, Isolated in a Laboratory.

Two novel Blastococcus sp. clones, TML/M2B and TML/C7B, with 2 different stable growth phenotypes, were isolated from a laboratory tissue culture. The draft genome sequences generated through genomic sequencing of clones TML/M2B and TML/C7B contain 4 and 2 contigs, respectively. The respective genome sizes are 4.10 Mb and 4.11 Mb, with G+C contents of 74.17% and 74.14%, respectively.

ANNOUNCEMENT

The genus Blastococcus, a member of the Geodermatophilaceae family, contains Gram-positive bacteria isolated mainly from soils, marine sediment, and stone interiors (1–3). In a rare case, an unclassified Blasctococcus sp. was isolated from a plant (4). The life cycle of Blastococcus spp. contains several cell forms, including motile rod and vibroid, as well as nonmotile cocci and coccoid aggregates (5). Here, we report the draft genome sequences of two clones of Blastococcus spp. isolated from a flask of cell culture in a laboratory.

In a prolonged laboratory tissue/cell culture experiment, we found that a flask of cell culture contained unusual microbes. The microorganisms grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (R10) were identified as Gram-positive bacteria (1, 5) and formed orange-pigmented colonies on the R10 agar plates. We performed single-colony picking and isolated a clone growing mostly as motile forms, designated TML/M2B, and a clone growing mostly as aggregated nonmotile coccoid forms, designated TML/C7B (microscopic observations). Genomic DNA of these two bacterial clones was prepared with DNAzol reagent (Thermo Fisher) using the manufacturer’s protocol designed for Gram-positive bacteria. Genomic DNA libraries were prepared with a Nextera DNA Flex library prep kit (Illumina) and subjected to genomic sequencing using an Illumina MiSeq instrument with 2 × 150-bp pair-ended sequencing mode. Raw reads were trimmed and assembled into 336 and 302 contigs using the Trim Reads and De Novo Assembly tools of CLC Genomics Workbench version 20.0.4. The blastn results of the assembled contigs revealed identical 16S rRNA sequences in these two clones that best matched that of Blastococcus saxobsidens strain DD2 (6, 7) in GenBank, with 99.8% identity.

To further improve the completeness of the draft genome sequences, we performed Oxford Nanopore MinION sequencing. Genomic DNA libraries were prepared using a rapid sequencing library preparation kit (SQK-RAD004; Oxford Nanopore Technologies), and sequencing was run using Flowcell version R9.4 (FLO-MIN106; Oxford Nanopore Technologies) for a 48-hour running time. Guppy version 3.2.8 was used to perform base calling to convert MinION FAST5 outputs to FASTQ files. The raw reads in the FASTQ files were assembled into contigs using the De Novo Assemble Long Reads tool of CLC Genomics Workbench version 20.0.4 with default settings. The de novo assembly generated 4 contigs for clone TML/M2B and 2 contigs for clone TML/C7B. The contigs were then polished using the paired-end reads of the respective clones generated from Illumina MiSeq sequencing to improve the correctness (Polish with Reads Tool, CLC Genomics Workbench version 20.0.4).

Whole-genome comparison (whole-genome alignment tools, CLC Genomics Workbench version 20.0.4) revealed that clones TML/M2B and TML/C7B had 86.45% average nucleotide identity (ANI) compared to Blastococcus saxobsidens strain DD2, and the ANI between these two clones was 99.98%. The genomes were submitted for comprehensive genome analysis using PATRIC version 3.6.8 (8) for annotation. Our findings are summarized in Table 1.

TABLE 1

Characterization of draft genome sequences of Blastococcus sp. clones TML/M2B and TML/C7B

Characteristic	Data for:
	Blastococcus sp. TML/M2B	Blastococcus sp. TML/C7B
Motility	Highly motile	Slightly motile
Morphology	Rod and vibroid dominant	Cocci and coccoid dominant
Contig count	4	2
No. of MiSeq reads	8,331,290	6,831,770
No. of minION reads	636,000	312,725
Draft genome length (bp)	4,114,186	4,100,268
N50 (bp)	1,784,222	2,703,711
G+C content (%)	74.17	74.14
Avg coverage (×)	230	134
BioProject accession no.	PRJNA685785	PRJNA685785
BioSample accession no.	SAMN17093989	SAMN17093990
SRA accession no.	SRR13283331, SRR13283332	SRR13283329, SRR13283330
GenBank Accession no.	JAENZZ000000000	JAEOAA000000000
PATRIC comprehensive genome analysis annotation
No. of annotated CDsa	4,071	4,036
No. of functional proteins	2,605	2,585
No. of hypothetical proteins	1,466	1,451
No. of annotated tRNAs	48	49
No. of annotated rRNAs	8	6
No. of repeat regions	74	46

CDs, coding sequences.

Data availability.

The raw reads and draft genome contigs of the clones Blastococcus sp. TML/M2B and TML/C7B have been deposited in the Sequence Read Archive (SRA) and GenBank under BioProject no. PRJNA685785, SRA no. SRR13283329, SRR13283330, SRR13283331, and SRR13283332, and GenBank accession no. JAENZZ000000000 and JAEOAA000000000.