Complete Genome Sequence of Rhodococcus sp. Strain W8901, a Human Clinical Specimen, Assembled Using MiSeq and MinION Sequence Data

ABSTRACT

Rhodococcus sp. strain W8901 is a Gram-positive, aerobic, mycolic acid-containing coccobacillus obtained from a patient with acute lymphocytic leukemia. Here, we report on the complete, circular genome sequence obtained using Illumina MiSeq and Oxford Nanopore Technologies MinION reads in order to better resolve the phylogeny of a rare pathogen.

ANNOUNCEMENT

The genus Rhodococcus (1) presently includes >50 validly named species (https://www.bacterio.net/genus/rhodococcus). Members of the genus Rhodococcus are characterized as Gram-positive, nonmotile, aerobic, catalase-positive, and non-endospore-producing, with their cell wall comprising chemotype IV peptidoglycan and mycolic acids composed of 34 to 64 carbon atoms (2–4). Rhodococcus species are noted for their catabolic and biodegradation abilities but, other than Rhodococcus equi, are rare opportunistic pathogens (3). Isolate 86-07, here designated strain W8901, was obtained by culturing central venous catheter blood from a patient diagnosed with acute lymphocytic leukemia. Isolate W8901 was streaked on Trypticase soy agar supplemented with 5% sheep blood (TSAB) and grown for 3 days at 35°C. As described by Langer et al. (5), genomic DNA from strain W8901 was purified and amplified by PCR, and subsequent multilocus sequence typing, and 16S rRNA gene sequence analysis showed close sequence similarity to R. equi DSM 20307^T. The complete genome of strain W8901 was sequenced to better understand the taxonomic assignment of this rare human pathogen.

Strain W8901 was streaked onto TSAB from stock maintained at the Centers for Disease Control and Prevention, and a colony was cultured in Trypticase soy broth for 3 days at 35°C at 200 rpm. Genomic DNA used for both libraries was purified according to the manufacturer’s protocol using the MasterPure DNA purification kit (Epicentre, Madison, WI). Genomic DNA libraries were made with the NEBNext Ultra DNA library prep kit (New England BioLabs, Ipswich, MA, USA) and the rapid barcoding kit (Oxford Nanopore Technologies [ONT]). Libraries sequenced with a 2 × 250-bp MiSeq (Illumina, San Diego, CA, USA) and a MinION (ONT) instrument using R9.4.1 flow cells produced 12,151,790 and 167,065 reads, respectively. Default parameters were used for all software unless otherwise specified. The long-read base calling with Guppy v3.2.8 generated an N₅₀ value of 14,417 bp. Flye v2.7-b1585 (6) was used to assemble a single circular 5,713,496-Mbp chromosome with 191× coverage. Visualization with Bandage v0.8.1 suggested that the genome is complete with a single circular chromosome (7). The MinION reads were then used to correct assembly errors with three sequential rounds of read mapping using Minimap v2.17-r941 with the “-x map-ont” setting and correction with Racon v1.3.2 (8, 9). Medaka’s consensus v1.0.1 (https://github.com/nanoporetech/medaka) function was used as a final long-read correction round prior to short-read (single nucleotide polymorphism [SNP] and indel) polishing. Paired short reads (trimmed to a Phred score of at least 30 with Trimmomatic v0.35 [10]) were mapped to the chromosome for sequential polishing stages until no more errors were identified. Unicycler’s polish function along with Bowtie v2.3.5.1, SAMtools v1.10, and Pilon v1.23 corrected 120 SNPs and 2,165 indels (11–14). Of the indels, 1,838 were homopolymers (98% were C or G), and the other remaining corrections were 37 insertions and 290 deletions. The dnaA sequence coordinates were located with Prokka v1.14.0, and the chromosome’s start was reoriented to it with Biopython v1.74 (15, 16). The polished chromosome contains 68.13% GC and was annotated with Prokaryotic Genome Annotation Pipeline (PGAP) v4.11 (17), which identified 5 rRNA gene operons and 5,062 putative protein-encoding genes. The current closest phylogenetically related genome is that of Rhodococcus defluvii Ca11^T with an ANI of 89.2% and digital DNA-DNA hybridization (dDDH) of 36.1%, which suggests that strain W8901 is within the radiation of the Rhodococcus genus (18, 19).

Data availability.

The whole-genome sequence of Rhodococcus sp. W8901 has been deposited at the DDBJ/ENA/GenBank database under the accession number CP054690. The version described in this paper is the first version, CP054690.1. The sequencing read data have been deposited in the NCBI SRA under accession numbers SRR11951435 and SRR11951436.