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The gold standard method for serotyping  Escherichia coli  has relied on antisera-based typing of the O- and H-antigens, which is labor intensive and often unreliable. In the post-genomic era, sequence-based assays are potentially faster to provide results, could combine O-serogrouping and H-typing in a single test, and could simultaneously screen for the presence of other genetic markers of interest such as virulence factors. Whole genome sequencing is one approach; however, this method has limited multiplexing capabilities, and only a small fraction of the sequence is informative for subtyping or identifying virulence potential. A targeted, sequence-based assay and accompanying software for data analysis would be a great improvement over the currently available methods for serotyping. The purpose of this study was to develop a high-throughput, molecular method for serotyping  E. coli  by sequencing the genes that are required for production of O- and H-antigens, as well as to develop software for data analysis and serotype identification. To expand the utility of the assay, targets for the virulence factors, Shiga toxins ( stx  1 , and  stx  2 ) and intimin ( eae ) were included. To validate the assay, genomic DNA was extracted from O-serogroup and H-type standard strains and from Shiga toxin-producing  E. coli , the targeted regions were amplified, and then sequencing libraries were prepared from the amplified products followed by sequencing of the libraries on the Ion S5™ sequencer. The resulting sequence files were analyzed  via  the SeroType Caller™ software for identification of O-serogroup, H-type, and presence of  stx  1  , stx  2 , and  eae . We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of  stx  1a,c,d  (3 of 3 strains),  stx  2c−e,g  (8 of 8 strains),  stx  2f  (1 strain), and  eae  (6 of 6 strains). Taken together, the high-throughput, sequence-based method presented here is a reliable alternative to antisera-based serotyping methods for  E. coli .

A Targeted Sequencing Assay for Serotyping Escherichia coli Using AgriSeq Technology