Closed genome sequences of two Listeria monocytogenes ST121 strains

ABSTRACT We performed Oxford Nanopore and Illumina sequencing to generate accurate, closed genomes for the Listeria monocytogenes strains 6179 and L58-55. The new assemblies were generally similar to the previous Illumina-based assemblies, but additional rRNA operons and repeat regions were identified in the new assembly for strain 6179.

severe infections and surviving antimicrobial conditions used to mitigate bacterial contamination in food production (1).Many L. monocytogenes strains belonging to sequence type (ST) 121 exhibit persistence, whereby a strain can be repeatedly isolated from the same food production environment over many years (2)(3)(4)(5).The L. monocy togenes ST121 strain 6179 was originally isolated from a cheese production facility in Ireland, and its response to stressors encountered in food production has been extensively studied (6)(7)(8)(9)(10)(11). Another ST121 strain, L58-55, was isolated from a human patient (12).Draft genomes for strains 6179 and L58-55 were previously generated using short-read sequencing (13,14).Here, we utilized long-and short-read sequencing to obtain closed genomes for each strain.
6179 and L58-55 were stored in tryptic soy broth (TSB) with 20% glycerol at −80°C.Strains were grown in TSB at 37°C to OD 600 ~2.5, and DNA was extracted using a Genomic-tip 20/G Kit (Qiagen).DNA samples were processed for library preparation without shearing and size selection using the SQK-LSK109 kit and the EXP-NBD104 barcoding kit; sequencing was conducted on an X5 flow cell using an Oxford Nanopore GridION with Guppy 4.3.4 for base-calling.Additionally, 6179 was sequenced on the Illumina MiSeq platform (250 bp, paired-end reads) after library preparation with the NEBNext Ultra II FS Kit.Available Illumina sequencing data from a previous MiSeq run (Illumina Nextera XT, 300 bp, paired-end reads; SRX16175649) was used for the L58-55 assembly (14).
For Illumina data, adapters were trimmed and low-quality reads were removed using BBDuk v37.36 with k, mink, and hdist values of 23, 11, and 1, respectively, with the tbo and tpe flags enabled.Quality trimming of both ends was performed with a minimum value of 20 (15).Hybrid assembly was then performed with processed Illumina data and raw long-read data in SPAdes 3.14.1 with the isolate option enabled (16).Contigs with k-mer coverage of less than 1 were discarded.Coverage statistics were calculated against the remaining contigs using the BBMap covstats option (15), and the circularity of the contigs was confirmed with Circlator v1.5.5 using default parameters (17).Genomes were annotated with PATRIC and the NCBI Prokaryotic Genome Annotation Pipeline (18)(19)(20).Percent nucleotide identities between the newly assembled contigs and the previous genomes were calculated using the NUCmer function of MUMmer 3.23 (21).
For both strains, sequencing and assembly yielded closed genomes consisting of two contigs, one representing the chromosome and one representing the plasmid.The average coverages, lengths, GC content, and sequencing statistics are shown in Table 1.The new assemblies were overall highly similar to the previous assemblies in terms of percent nucleotide identity (Table 1).However, the hybrid 6179 chromosome assembly is 26,072 bp longer than the previous version.This discrepancy was primarily due to two previously undetected rRNA operons and to multiple repeat regions primarily located within prophages (Table 1).This work adds two high-quality assemblies to the list of L. monocytogenes genomes.

TABLE 1
Statistics for the chromosome and plasmid assemblies of L. monocytogenes strains 6179 and L58-55 The previous assemblies were based on Illumina sequencing only, and the genomes were not closed. a