Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0317-z) contains supplementary material, which is available to authorized users.

Metagenomic Virome Sequencing in Living Donor and Recipient Kidney Transplant Pairs Revealed JC Polyomavirus Transmission

“…Metagenomic sequencing and data analysis was performed as described previously [15,16] (Supplementary Methods). Briefly, samples were centrifuged, filtered (0.45 μm), and nuclease-treated.…”

Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples

“…Numerous protocols for viral enrichment and genome amplification have been described in literature [5][6][7]. Commonly employed protocols such as sample filtration [8], nuclease digestion, ultracentrifugation, and random pre-amplification of RNA or DNA in separate reactions would be particularly useful for increasing the signal-to-noise ratio in the viral analysis of biological samples in which the levels of nucleic acid background are high.…”

Correction to: Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

“…Following publication of the original article [1], the authors were alerted by a colleague of a column duplication in Table 1. Since the summary row was correct, though, the interpretation and the conclusion of the article were not affected…”