ColiSeq: a multiplex amplicon assay that provides strain level resolution of Escherichia coli directly from clinical specimens

Charles H.D. Williamson, Adam J. Vazquez, Amalee E. Nunnally, Kristen Kyger, Viacheslav Y. Fofanov, Tara N. Furstenau, Heidie M. Hornstra, Joel Terriquez, Paul Keim, Jason W. Sahl

Research output: Contribution to journalArticlepeer-review


Escherichia coli is a diverse pathogen, causing a range of disease in humans, from self-limiting diarrhea to urinary tract infections (UTIs). Uropathogenic E. coli (UPEC) is the most frequently observed uropathogen in UTIs, a common disease in high-income countries, incurring billions of dollars yearly in treatment costs. Although E. coli is easily grown and identifiedin the clinical laboratory, genotyping the pathogen is more complicated, yet critical for reducing the incidence of disease. These goals can be achieved through whole-genome sequencing of E. coli isolates, but this approach is relatively slow and typically requires culturing the pathogen in the laboratory. To genotype E. coli rapidly and inexpensively directly from clinical samples, including but not limited to urine, we developed and validated a multiplex amplicon sequencing assay, called ColiSeq. The assay consists of targets designed for E. coli species confirmation,high resolution genotyping, and mixture deconvolution. To demonstrate its utility, we screened the ColiSeq assay against 230 clinical urine samples collected from a hospital system in Flagstaff,Arizona, USA. A limit of detection analysis demonstrated the ability of ColiSeq to identify E. coli at a concentration of ∼2 genomic equivalent (GEs)/mL and to generate high-resolution genotyping at a concentration of 1 × 105 GEs/mL. The results of this study suggest that ColiSeq could be a valuable method to understand the source of UPEC strains and guide infection mitigation efforts.As sequence-based diagnostics become accepted in the clinical laboratory, workflowssuch as ColiSeq will provide actionable information to improve patient outcomes.

Original languageEnglish (US)
JournalMicrobiology spectrum
Issue number6
StatePublished - Jun 2024


  • amplicon sequencing
  • E. coli
  • genotyping
  • UTIs

ASJC Scopus subject areas

  • Physiology
  • Ecology
  • General Immunology and Microbiology
  • Genetics
  • Microbiology (medical)
  • Cell Biology
  • Infectious Diseases


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