Complete Genome Sequence of a Virulent Leptospira interrogans Serovar Copenhageni Strain, Assembled with a Combination of Nanopore and Illumina Reads.

Here, we present the complete genome sequence of a highly virulent Leptospira interrogans serovar Copenhageni strain isolated from a dog with severe leptospirosis. In this work, a gapless genome draft was assembled with a combination of Nanopore and Illumina data of relatively low coverage.

ANNOUNCEMENT

Leptospira species are spirochete bacteria that colonize proximal renal tubules of mammals, and infections may cause fatal disease in humans and animals. In this study, we assembled the genome of Leptospira interrogans strain SK1 and compared it with those of L. interrogans serovar Copenhageni (strain Fiocruz L1-130) (1) and L. interrogans serovar Lai (strain 56601) (2). The SK1 strain was isolated from the blood of a canine patient that succumbed to severe clinical leptospirosis (3). The strain was isolated through routine Leptospira culture protocols using Ellinghausen-McCullough-Johnson-Harris medium, as described previously (1).

Genomic DNA from strain SK1 was isolated with the MasterPure complete DNA and RNA purification kit (Epicentre, USA). Sequencing libraries for the MiSeq and MinION platforms were prepared with the Nextera XT DNA library preparation kit (Illumina, USA) and the ligation sequencing kit (SQK-LSK109; Oxford Nanopore Technologies, UK), respectively, following the manufacturers’ instructions. Quality control was performed with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc). In the case of Nanopore reads, we used Guppy v.3.2.4 for base calling and Porechop v.0.2.4 (https://github.com/rrwick/Porechop) for adapter trimming. De novo assembly was performed with Canu v.2.0 (4) using only the Nanopore reads. The Canu assembly was initially polished with Medaka v.0.11.4 (https://nanoporetech.github.io/medaka) using probabilistic model r941_min_fast_g303. Pilon v.1.23 (5) was used for hybrid polishing with the Illumina reads. To estimate the number of sequencing errors after each polishing step, Illumina reads were aligned to the Nanopore assembly with BWA v.0.7.12 (6). BCFtools v.1.9 (7) was then used to call for single-nucleotide variants and insertion/deletions (indels), which were considered to be errors if their mapping quality score was above 20. RATT was used to transfer the genes annotated in the Fiocruz L1-130 genome to the SK1 assembly. Prokka v.1.14.3 (8) was used to complement the RATT results. Default parameters were used for all software except where otherwise noted.

Illumina and Nanopore sequencing generated 1.1 million 300-bp paired-end reads and 142,300 reads with a median length of 8 kb, respectively. De novo assembly of Nanopore reads resulted in 2 contigs, consistent with the 2 Leptospira chromosomes. The number of suspected errors decreased from 14,672 (0.3%) in the original Canu assembly to 92 (0.002%) after polishing with Pilon. Of these, only 6 errors seemed to affect putative coding sequences and were corrected manually.

The SK1 genome is very similar to that of strain Fiocruz L1-130 in terms of synteny (Fig. 1) and global metrics, including a total size of 4,630,180 bp, a G+C content of 35%, and 3,731 protein-coding genes. However, at least 11 pseudogenes from Fiocruz L1-130 appear to have functional orthologs in the genomes of strains SK1 and 56601, despite the latter belonging to L. interrogans serovar Lai. Conversely, genes folA and folC, encoding dihydrofolate reductase and dihydrofolate synthase, respectively, appear to have become pseudogenes in SK1, which suggests impaired folate metabolism in this strain.

Fig 1

Comparison of the genomes of L. interrogans serovar Copenhageni strains SK1 and Fiocruz L1-130. Pink bands represent regions of shared sequence similarity. Coverage of Nanopore and Illumina reads is plotted along the SK1 genome, averaged over a 500-bp sliding window. Chr, chromosome.

Data availability.

The genome sequence of the SK1 strain and associated data were deposited under BioProject number PRJNA600490, GenBank accession numbers CP048830 (chromosome I) and CP048831 (chromosome II), and SRA accession numbers SRX7657977 (Illumina reads) and SRX7730232 (MinION reads).