Application of Metagenomic Long-Read Sequencing for the Diagnosis of Herpetic Uveitis

Abstract

Purpose: To investigate the sensitivity and specificity of herpes virus detection by nanopore metagenomic analysis (NMA) compared with multiplex polymerase chain reaction (mPCR)-positive and -negative controls.

Methods: This study included 43 patients with uveitis who had been screened for intraocular herpes virus infection using mPCR from aqueous humor samples. Aqueous humor samples stored after mPCR were subjected to whole-genome amplification, long-read sequencing, and analysis of the phylogenetic microorganism composition using a Flongle flow cell on the Oxford Nanopore MinION platform. For samples that tested positive with mPCR and negative with the Flongle flow cell, additional long-read sequencing was performed using a MinION flow cell, which enabled acquisition of more sequence data. The sensitivity and specificity of herpes virus detection by NMA were compared with the mPCR-positive and -negative controls.

Results: NMA using a Flongle flow cell detected the pathogenic virus in 60.0% of those who tested positive by mPCR (12/20). Further analysis using the MinION flow cell successfully identified viral DNA fragments in three out of the eight initially undetected samples, yielding a collective sensitivity of 75.0% (15/20). All of the virus detected with the long-read sequencing were identical to those diagnosed by mPCR testing, and none of the samples that tested negative by mPCR revealed herpes viral DNA with the use of long-read sequencing.

Conclusions: For the detection of etiologic herpes virus DNA fragments, NMA revealed a reasonable sensitivity and high specificity. Our study highlights the potential of nanopore sequencing to facilitate further advances in uveitis diagnosis.

Figure 1.

Phylogenetic classification of a representative mPCR-positive uveitis specimen (P5), time-point analysis of the number of sequence reads, and visualization of genomic data. (A) Analysis results of case with CMV retinitis (P5), a representative of herpes virus uveitis. The phylogenetic tree (Pavian plot) shows all microorganisms, except the human genome, and quantifies them from left to right in the following order: domain (D), phylum (P), class (C), family (F), genus (G), and species (S). (B) Time-point analysis of the number of sequence reads mapped to the CMV reference genome. The horizontal axis indicates time (minutes), and the vertical axis indicates the number of sequence reads mapped to the reference genome of CMV. CMV DNA reads were detected approximately 30 minutes after sequencing initiation. (C) The reads of virus-derived DNA fragments were mapped onto the CMV reference genome and visualized with Integrative Genomics Viewer (IGV). Panel C was created using data at the end of the run in Flongle sequencing (i.e., at 24 hours).

Figure 2.

Comparison of Flongle flow cell and MinION flow cell nanopore metagenomic analyses of an mPCR-positive uveitis specimen (P11). To determine the reason for the low sensitivity of the nanopore metagenomic analysis, we used a MinION flow cell to test whether the amount of sequence data affected detection sensitivity in mPCR-positive cases that were negative with Flongle flow cell sequencing. The MinION flow cell produced around 10 times more sequence data than the Flongle flow cell, and two CMV reads were detected.

Figure 3.

Proportions of Homo sapiens, bacterial, archaeal, and viral reads in the output data of nanopore metagenomic analyses of mPCR-positive uveitis specimen. From the 20 specimens positive for the herpes virus with mPCR, we examined the nanopore sequence data origin ratios. In addition to bacteria, viruses, and archaea, the human genome and unclassified nanopore sequence reads were also detected. Samples in which the etiologic virus was detected in the Flongle flow cell or the MinION flow cell are identified by a plus sign (+) in the figure, and samples in which it was not detected are marked in the bottom row with a minus sign (−).