Metagenomic profiling of long-read sequencing for clinical diagnosis of ocular inflammation

Objective To establish a metagenomic profiling method using long-read sequencing for clinical diagnosis of ocular inflammation and detect the etiologic virus of herpetic uveitis. Design A retrospective, cross-sectional study. Participants The participants were 44 uveitis patients with a suspected infectious etiology and 22 controls with cataract. Methods The anterior aqueous humor (10-20 µl) was subjected to DNA purification, followed by whole genome amplification. The Nanopore MinION™ using the Flongle Flow Cell was used to perform rapid long-read sequencing and the phylogenetic composition of the microorganisms in the specimen was evaluated. Main Outcomes and Measures The detection of the DNA sequence reads of the etiologic virus of herpetic uveitis in the generated FASTQ files from nanopore sequencing and the evaluation of the limits of detection (LOD) of metagenomic analysis compared to multiplex polymerase chain reaction (mPCR) testing for etiologic virus detection of herpetic uveitis. Results The detection rate of nanopore metagenomic analysis was approximately 59.0% as a result of validation against 22 mPCR-positive cases. The LOD was between 10 3.6 and 10 6 copies of virus DNA. The undetectable cases tended to have significantly lower copy numbers by mPCR, suggesting the lower metagenomic analysis sensitivity compared to mPCR. The nine pathogenic microorganisms evaluated by mPCR were also not detected by nanopore in all mPCR-negative cases and controls. The minimum time to obtain analysis results using this method was approximately 190 minutes. Conclusions and Relevance Our established sequencing protocol from the anterior aqueous humor detected the DNA fragments of etiologic viruses in patients with herpes virus uveitis. Conversely, nanopore metagenomic results contained considerable noise and were found to be less sensitive compared to the conventional mPCR tests for ocular infections.