Complete Genome Sequence of Rhodococcus sp. Strain M8, a Platform Strain for Acrylic Monomer Production

We report a 6.27-Mbp complete genome of Rhodococcus sp. strain M8, an originally discovered strain that is now under investigation for production of acrylic monomers. The genome consists of a 6.1-Mbp circular chromosome and a 173.2-kbp plasmid.

ABSTRACT

We report a 6.27-Mbp complete genome of Rhodococcus sp. strain M8, an originally discovered strain that is now under investigation for production of acrylic monomers. The genome consists of a 6.1-Mbp circular chromosome and a 173.2-kbp plasmid.

ANNOUNCEMENT

Here, we present a complete sequence of the Rhodococcus rhodochrous strain M8 genome, which was obtained after Nanopore de novo sequencing and joining of this sequence with the previously obtained draft genome sequence of M8 (1). The effort to close this genome was undertaken because the strain is actually a platform for the development of biocatalysts for acrylic monomer production (several biocatalysts were derived from it earlier; see references 2–4 and also see the information about biocatalytic production of acrylic monomers [5, 6]). Additionally, the strain is one of the model strains for basic research on the genetics of rhodococci (1, 7–9). Further basic and applied research based on the strain requires precise genome information. Together with that, rhodococcal genomes are significantly variable in whole-genome size and the presence of genes for secondary metabolism. The quantity of complete rhodococcal genomes is too low now (five genomes [10]); therefore, this new genome is a valuable contribution to the knowledge base in this field.

The genomic DNA was extracted from a culture grown in Luria broth and purified using the Puregene Yeast/Bact. kit B (Qiagen), and PCR-free libraries were made with a ligation sequencing kit (SQK-LSK109), according to the manufacturer’s protocol for native barcoding of genomic DNA. The libraries were sequenced using an Oxford Nanopore Technologies (ONT) platform with a MinION R9 flow cell by Genotek (Moscow, Russia), and FAST5 files were generated. Base calling was performed on a local system using ONT Guppy software v3.4.1 with GPU support (https://github.com/gnetsanet/ONT-GUPPY), with default parameters. The resulting FASTQ file was used for adapter trimming with Porechop v0.2.4 (https://github.com/rrwick/Porechop) with default parameters. After that, 133,682 reads were obtained, with an N₅₀ value of 12.3 kbp. Low-quality nucleotides and short reads were removed using Cutadapt v2.7 (11) with the parameters -q 20 -m 1000. Thus, 93,190 high-quality reads were obtained. These reads were corrected using Canu v1.7 (12) with the parameters -correct -nanopore genomeSize = 6000k. The corrected reads obtained had an average length of 8,382 nucleotides (nt) and a maximal length of 77,355 nt. These reads were used for de novo genome assembly using Flye v2.7 (13) with default parameters, resulting in two contigs of 6.1 and 0.17 Mbp. Finally, Illumina reads obtained previously (1) (also see the link to the SRA record below) were mapped to the Nanopore assembly using BWA MEM v0.7.15 (14) with default parameters. The BAM file obtained was used for genome polishing with Pilon v1.23 (15) with default parameters. After these procedures, the two contigs mentioned above remained separate DNAs. These 6.1- and 0.17-Mbp contigs were checked for circularity using Circlator (16), with default parameters. As a result, the 6.1-Mbp contig was shown to be a circular 6,106,346-nt chromosome, while the 0.17-Mbp contig remained not closed to the circle. The latter was also checked manually for the absence of overlapping ends. We suggest that the 0.17-Mbp contig could be a linear 173,200-nt plasmid. The average coverage for both contigs was estimated as 236-fold, and the complete genome was not rotated to any certain base. Finally, we checked that 93% of all Nanopore reads and 95% of all Illumina reads were mapped on the completed genome. Gene predictions and annotations were performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (17), and 5,708 genes, including 5,639 coding sequences, were predicted.

Data availability.

This genome sequence has been deposited in NCBI GenBank with accession number GCA_015654185. Raw sequencing reads used in the work are available under SRX9609528 (Nanopore) and SRR13296353 (Illumina). The version of the complete genome described in this paper is the first version, GCA_015654185.1.