Current infectious disease molecular tests are largely pathogen specific, requiring test selection based on the patient’s symptoms. For many syndromes caused by a large number of viral, bacterial, or fungal pathogens, such as respiratory tract infections, this necessitates large panels of tests and has limited yield. In contrast, next-generation sequencing-based metagenomics can be used for unbiased detection of any expected or unexpected pathogen. However, barriers for its diagnostic implementation include incomplete understanding of analytical performance and complexity of sequence data analysis.
Researchers at the University of Utah School of Medicine compared detection of known respiratory virus-positive (n= 42) and unselected (n= 67) pediatric nasopharyngeal swabs using an RNA sequencing (RNA-seq)-based metagenomics approach and Taxonomer, an ultrarapid, interactive, web-based metagenomics data analysis tool, with an FDA-cleared respiratory virus panel (RVP; GenMark eSensor). Untargeted metagenomics detected 86% of known respiratory virus infections, and additional PCR testing confirmed RVP results for only 2 (33%) of the discordant samples. In unselected samples, untargeted metagenomics had excellent agreement with the RVP (93%). In addition, untargeted metagenomics detected an additional 12 viruses that were either not targeted by the RVP or missed due to highly divergent genome sequences. Normalized viral read counts for untargeted metagenomics correlated with viral burden determined by quantitative PCR and showed high intrarun and interrun reproducibility. Partial or full-length viral genome sequences were generated in 86% of RNA-seq-positive samples, allowing assessment of antiviral resistance, strain-level typing, and phylogenetic relatedness.
Respiratory virus detection by untargeted metagenomics and RVP
(A) Fractional abundance of human, bacterial, and viral sequences in untargeted metagenomics data from an influenza A virus-positive NP swab of a female infant determined by Taxonomer. Approximately 1.5% of reads were of viral and 2.7% of bacterial origin. (B) Of 1.74 × 105 viral reads, 1.73 × 105 (99.4%) could be classified to the species level (influenza A virus) and 7.9 × 104 (45.3%) to the H1N1 subtype. (C) Untargeted metagenomics identified 36 of 42 (86%) respiratory viruses detected by the RVP. Four of the 6 viruses missed by untargeted metagenomics (blue bars) could not be detected by qPCR (hashed bars), resulting in detection of 36 of 38 viruses (95%) that were consistently detected by targeted methods. (D) Untargeted metagenomics detected more viral infections (n = 48, including 11 not targeted by the RVP [asterisk]) than the RVP (n = 37) in unselected NP swabs (n = 67) collected during a 12-month period. ADV, adenovirus; HMPV, human metapneumovirus; IAV, influenza A virus; PIV, parainfluenza virus; HRV, human rhinovirus; RSV, respiratory syncytial virus; HCoV, human coronavirus; CMV, cytomegalovirus; HBoV, human bocavirus; EV, enterovirus; MV, measles virus.
Overall, untargeted metagenomics had high agreement with a sensitive RVP, detected viruses not targeted by the RVP, and yielded epidemiologically and clinically valuable sequence information.