Purpose: The purpose of this study was to evaluate next generation sequencing technology as an applicable subtyping tool for compliance inspection of L. monocytogenes contamination for smoked fish-processing facilities and food recalls.
Methods: Sequencing genomic libraries of L. monocytogenes isolates derived from five smoked fish-processing facilities over ten years were prepared using the Illumina Nextera XT kit and subsequently applied for 2×250 bp paired-end sequencing runs on the Illumina MiSeq sequencer. The raw data of fastq files were imported as paired reads into the Qiagen CLC Genomics Workbench for further data analysis and SNP-based phylogenetic tree construction.
Results: We have successfully sequenced 71 L. monocytogenes genomes from those fish-processing facilities using the Illumina MiSeq sequencer. By using K-mer based spectra, 7 best matched references derived from NCBI genome database were yielded for those 71 genomes, among which 35% (25/71), 25% (18/71) and 30% (21/71) of L. monocytogenes genomes matched to 3 core references. K-mer based phylogenetic analysis revealed NZ_HG813249 as the common reference for further analysis. We were able to yield average coverages of 120±34 for sequencing depth, (95.3±2.4)% of mapped reads and 64,832±56,355 high quality variants (range 198-254,281) when mapping reads to the common reference. SNP-based phylogenetic analysis revealed 14 clades resulting in 3 large ones containing 20, 16 and 18 genomes, respectively.
Significance: Our data indicate that next generation sequencing is a valuable subtyping tool for analyzing L. monocytogenes isolates from smoked fish firms.