Abstract
BackgroundSequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnostics. Here, we describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples.ResultsIn order to optimize metagenomic sequencing for application in virus diagnostics, we tested different enrichment and amplification procedures on plasma samples spiked with RNA and DNA viruses. A protocol including filtration, nuclease digestion, and random amplification of RNA and DNA in separate reactions provided the best results, allowing reliable recovery of viral genomes and a good correlation of the relative number of sequencing reads with the virus input. We further validated our method by sequencing a multiplexed viral pathogen reagent containing a range of human viruses from different virus families. Our method proved successful in detecting the majority of the included viruses with high read numbers and compared well to other protocols in the field validated against the same reference reagent. Our sequencing protocol does work not only with plasma but also with other clinical samples such as urine and throat swabs.ConclusionsThe workflow for virus metagenomic sequencing that we established proved successful in detecting a variety of viruses in different clinical samples. Our protocol supplements existing virus-specific detection strategies providing opportunities to identify atypical and novel viruses commonly not accounted for in routine diagnostic panels.
Highlights
Sequence-specific PCR is the most common approach for virus identification in diagnostic laboratories
Despite the numerous promising attempts to apply metagenomics to virology, direct sequencing of nucleic acids obtained from biological samples results in a high background of genetic material mainly derived from the host and bacteria hampering the detection of viruses [22, 23]
Plasma from healthy donors was spiked with different viruses and extracted, and the concentration in the eluate determined by quantitative real-time PCR (qPCR)
Summary
Sequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. We describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples. Sequence-specific PCR is the most common approach for virus identification and quantification in diagnostic laboratories as it is highly sensitive, rapid, and cost effective. Despite the numerous promising attempts to apply metagenomics to virology, direct sequencing of nucleic acids obtained from biological samples results in a high background of genetic material mainly derived from the host and bacteria hampering the detection of viruses [22, 23]. Sample type greatly influences the composition of sequencing reads, and due to the complexity of clinical materials, sample preparation and virus enrichment methods need to be adapted. The anchored random PCR approach, as used in our study, has been frequently used and described in detail in previous studies [16, 36,37,38,39,40,41,42]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
