DNA sequencing and other DNA-based methods are now broadly used for detection and identification of bacterial foodborne pathogens. For the identification of foodborne bacterial pathogens, taxonomic assignments must be made to the species or even subspecies level. Long-read DNA sequencing provides finer taxonomic resolution than short-read sequencing. Here, we demonstrate the potential of long-read shotgun sequencing obtained from the Oxford Nanopore Technologies (ONT) MinION single-molecule sequencer, in combination with the Basic Local Alignment Search Tool (BLAST) with custom sequence databases, for foodborne pathogen identification. A library of mixed DNA from strains of the “Super-7” Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157[:H7]) was sequenced using the ONT MinION resulting in 44,245 long-read sequences. The ONT MinION sequences were compared to a custom database composed of the E. coli O-antigen gene clusters. A vast majority of the sequence reads were from outside of the O-antigen cluster and did not align to any sequences in the O-antigen database. However, 58 sequences (0.13% of the total sequence reads) did align to a specific Super-7 O-antigen gene cluster, with each O-antigen cluster aligning to at least four sequence reads. BLAST analysis against a custom whole-genome database revealed that 5096 (11.5%) of the MinION sequence reads aligned to one and only one sequence in the database, of which 99.6% aligned to a sequence from a “Super-7” STEC. These results demonstrate the ability of the method to resolve STEC to the serogroup level and the potential general utility of the MinION for the detection and typing of foodborne pathogens.

DNA测序和其他基于DNA的方法现已广泛用于细菌性食源性病原体的检测和鉴定。为了鉴定食源性细菌病原体，必须对物种甚至亚种级别进行分类分配。与短读测序相比，长读DNA测序可提供更好的分类分辨率。在这里，我们展示了从牛津纳米孔技术（ONT）MinION单分子测序仪与基础局部比对搜索工具（BLAST）与自定义序列数据库相结合获得的长读shot弹枪测序的潜力，可用于食源性病原体鉴定。来自“ Super-7”滋贺菌毒素大肠杆菌菌株的混合DNA文库（STEC）血清群（O26，O45，O103，O111，O121，O145和O157 [：H7]）进行了测序，得出了44,245个长读序列。将ONT MinION序列与由大肠杆菌组成的自定义数据库进行比较O-抗原基因簇。绝大多数序列读数来自O-抗原簇的外部，并且与O-抗原数据库中的任何序列都不比对。但是，有58个序列（占总序列读数的0.13％）确实与特定的Super-7 O-抗原基因簇比对，每个O-抗原簇与至少四个序列读数比对。针对定制的全基因组数据库的BLAST分析显示，有5096个（11.5％）MinION序列与数据库中的一个序列和一个序列比对，其中99.6％与“ Super-7” STEC的序列比对。这些结果证明了该方法具有将STEC解析到血清群水平的能力，以及MinION在检测和分类食源性病原体方面的潜在通用性。