Broad-Range Virus Detection and Discovery Using Microfluidic PCR Coupled with High-throughput Sequencing

Abstract

There is a need for a comprehensive and sensitive method to test for a broad range of viral pathogens in samples without any identifiable pathogen detected. Real-time PCR assays are sensitive and rapid, but their specificity limits their utility in detecting divergent agents. Shotgun high-throughput sequencing methods provide unbiased sequence identification, however, they have limited sensitivity and require complex analyses. In order to meet the need for a sensitive, high-throughput virus detection and discovery platform with good sensitivity, we combine two existing technologies, broadly-reactive consensus-degenerate pan-viral group PCR and the MiSeq sequencer (Illumina), using the Access Array (Fluidigm), a commercially-available microfluidic PCR system. Pan-viral group primers target conserved regions of virus taxonomic groups and can amplify known and potentially novel species. The Access Array employs dozens of these assays in parallel, which are then sequenced all at once on the MiSeq. In this study, we run a respiratory panel of pan-viral group PCR assays using AA-PCR-Seq. We validate the panel on a collection of representative human and animal samples, comparing it to qPCR and shotgun next-generation sequencing (NGS). AA-PCR-Seq provides a robust, straightforward method for screening large numbers of samples for virus detection and discovery.