Seqsphere v9

Whole genome sequencing was conducted at Omega Bioservices (Norcross, GA, USA). Briefly, DNA was extracted using the E.Z.N.A.^® Bacterial DNA Kit (Omega Bio-tek, Norcross, GA, USA). The concentration was measured using the QuantiFluor dsDNA System on a Quantus Fluorometer (Promega, Madison, WI, USA). A Kapa Biosystems HyperPlus kit (Kapa Biosystems, Wilmington, MA, USA) was used for the whole-genome library construction. DNA was fragmented, and ends were repaired, 3’ adenylated, and ligated to adapters. The resulting adapter-ligated libraries were PCR-amplified. After Illumina indexes were added, they were pooled for multiplexed sequencing on an Illumina X10 platform (Illumina, San Diego, CA, USA) using the pair-end 150 bp run format. de novo assembly was performed on Ridom SeqSphere + v9.0 (Ridom GmbH, Germany) using SKESA 2.4.0 (Souvorov et al., 2018 (link)).

In silico MLST was performed to identify clonal complex (CC) and sequence type (ST) on CLC genomic workbench (Qiagen, Redwood City, CA, USA) using seven housekeeping genes of L. monocytogenes, including ABC transporter (abcZ), beta-glucosidase (bglA), catalase (cat), succinyl diaminopimelate dessucinylase (dapE), d-amino acid aminotransferase (dat), l-lactate dehydrogenase (ldh), and histidine kinase (lhkA). An allele number was assigned to the sequence of each allele and CC determined based on the definition of Ragon et al. (2008) (link) and the Pasteur MLST database.¹ WGS data were further subjected to cgMLST cluster identification on Ridom SeqSphere + v9.0 (Ridom GmbH, Germany) (L. monocytogenes cgMLST scheme, 1701 loci) (Ruppitsch et al., 2015 (link)). The cluster distance threshold for the core genome was 10 allele differences. Epidemic clones of ECI, ECII, ECIII and ECIV (Chen and Knabel, 2007 (link)) were downloaded from the NCBI website.