Agnostic Sequencing for Detection of Viral Pathogens

Abstract

The advent of next-generation sequencing (NGS) technologies has expanded our ability to detect and analyze microbial genomes and has yielded novel molecular approaches for infectious disease diagnostics. While several targeted multiplex PCR and NGS-based assays have been widely used in public health settings in recent years, these targeted approaches are limited in that they still rely on a priori knowledge of a pathogen's genome, and an untargeted or unknown pathogen will not be detected. Recent public health crises have emphasized the need to prepare for a wide and rapid deployment of an agnostic diagnostic assay at the start of an outbreak to ensure an effective response to emerging viral pathogens. Metagenomic techniques can nonspecifically sequence all detectable nucleic acids in a sample and therefore do not rely on prior knowledge of a pathogen's genome. While this technology has been reviewed for bacterial diagnostics and adopted in research settings for the detection and characterization of viruses, viral metagenomics has yet to be widely deployed as a diagnostic tool in clinical laboratories. In this review, we highlight recent improvements to the performance of metagenomic viral sequencing, the current applications of metagenomic sequencing in clinical laboratories, as well as the challenges that impede the widespread adoption of this technology.

Keywords: SARS-CoV-2; diagnostic; metagenomic; molecular epidemiology; next-generation sequencing; respiratory viruses; viral diagnostics.

FIG 1

Summary of nonmolecular and traditional methods for molecular diagnosis and characterization of viral pathogens (top panels), along with more detailed workflows for molecular diagnostic techniques (bottom panels).

FIG 2

Sample preparation, library preparation, sequencing, and data analysis workflows for both targeted (i.e., amplicon NGS) (A) and untargeted (i.e., mNGS) (B) for detection of viral pathogens. The basic workflow for mNGS and amplicon sequencing is often similar until the cDNA synthesis and PCR step, where amplified mNGS approaches are characterized by random amplification techniques rather than targeted multiplex PCR. mNGS approaches may also be unbiased, as cDNA is directly sequenced without amplification to avoid amplification bias. Unbiased mNGS is difficult for samples with low abundance of viral nucleic acid or high abundance of host nucleic acid. mNGS often requires deeper sequencing for detection of viral pathogens. Detection of coinfections and unknown pathogens is possible with mNGS but not amplicon sequencing.

FIG 3

(A) Model of initial upper respiratory specimen collection and processing workflow detailing the nucleic acid composition of many clinical samples. Viral nucleic acid often represents a very small proportion of the total nucleic acid within a sample. (B) Schematics of approaches for pathogen enrichment (i to iii) and host depletion (iv and v) that have been utilized in mNGS workflows. Pathogen-enriched primers-probes are highlighted in red. Pathogen enrichment approaches include the following: (i) not-so-random primers that do not bind to off-target nucleic acid; (ii) metagenomic sequencing with spiked primer enrichment (MSSPE), in which primers for targeted pathogens are spiked at higher concentrations; and (iii) capture probe enrichment, where hybridized fragments are enriched following a magnetic bead pulldown. (iv) The host depletion approaches are illustrated. Depletion of abundant sequences by hybridization (DASH) (iv), and versus ONT’s adaptive sampling (v).