Patients (n = 2) presenting to the Birmingham and Midland Eye Centre, Birmingham, West Midlands, UK with suspected microbial keratitis affecting one eye were recruited to the study. Informed written consent was obtained. The research followed the tenets of the Declaration of Helsinki and was approved by the Health Research Authority Ethics Committee (Rapid Diagnosis of Ocular Infections (RADAR); Reference: 11/EM/0274).

Standard clinical microbiology corneal scrape culture results were compared with nanopore sequencing. For each patient, corneal scrapes were taken for standard clinical microbiology as per routine clinical practice (Lin et al., 2019). Corneal scrapes were taken at the base and edge of the lesion, inoculated onto blood, chocolate and Sabaraud’s agar plates, and processed at the local clinical microbiology laboratory (The agar plates were incubated for 5 days—blood and chocolate (6% CO2 at 37 °C); Sabourand’s (air at 30 °C)). The collection method with the highest DNA recovery in the ex vivo porcine study was used to collect corneal, conjunctival and negative control samples from the patients. Swabs were taken from the affected cornea and unaffected contralateral conjunctiva, together with a negative control ‘air swab’ of the examination room at the time point of participant sampling, to exclude contamination. Swabs were placed immediately into a ZR BashingBead Lysis Tube containing 750 ul of DNA Shield (Zymo Research, Irvine, CA, USA) and stored at −80 °C until DNA extraction, as described above. Sequence data were compared with the corresponding microbiology culture results of the hospital corneal scrapes. In cases where there was no concordance between culture and nanopore sequencing results, Illumina MiSeq 16S rRNA V4 sequencing was performed.

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