Abstract
BackgroundSequencing-based detection and characterization of viruses in complex samples can suffer from lack of sensitivity due to a variety of factors including, but not limited to, low titer, small genome size, and contribution of host or environmental nucleic acids. Hybridization-based target enrichment is one potential method for increasing the sensitivity of viral detection via high-throughput sequencing.ResultsThis study expands upon two previously developed panels of virus enrichment probes (for filoviruses and for respiratory viruses) to include other viruses of biodefense and/or biosurveillance concern to the U.S. Department of Defense and various international public health agencies. The newly expanded and combined panel is tested using carefully constructed synthetic metagenomic samples that contain clinically relevant amounts of viral genetic material. Target enrichment results in a dramatic increase in sensitivity for virus detection as compared to shotgun sequencing, yielding full, deeply covered viral genomes from materials with Ct values suggesting that amplicon sequencing would be likely to fail. Increased pooling to improve cost- and time-effectiveness does not negatively affect the ability to obtain full-length viral genomes, even in the case of co-infections, although as expected, it does decrease depth of coverage.ConclusionsHybridization-based target enrichment is an effective solution to obtain full-length viral genomes for samples from which virus detection would fail via unbiased, shotgun sequencing or even via amplicon sequencing. As the development and testing of probe sets for viral target enrichment expands and continues, the application of this technique, in conjunction with deeper pooling strategies, could make high-throughput sequencing more economical for routine use in biosurveillance, biodefense and outbreak investigations.
Highlights
Sequencing-based detection and characterization of viruses in complex samples can suffer from lack of sensitivity due to a variety of factors including, but not limited to, low titer, small genome size, and contribution of host or environmental nucleic acids
To expand the range of viruses that could be detected, in this study those two probe panels were combined with new probes for 41 additional viruses that are of biosurveillance and biodefense concern, for a full panel targeting 83 diverse viruses (Table 1)
In order to test this newly expanded probe panel and to assess the effect of hybridization-based viral enrichment on the sensitivity of High-Throughput Sequencing (HTS) for detection of a single virus within a complex environmental sample, commercial bat guano was spiked with increasing concentrations of Influenza virus (IFV)
Summary
Sequencing-based detection and characterization of viruses in complex samples can suffer from lack of sensitivity due to a variety of factors including, but not limited to, low titer, small genome size, and contribution of host or environmental nucleic acids. Detection via HTS is much less susceptible to false-negative results caused by antigenic drift or signature erosion. Despite these advantages, one of the technical challenges encountered with respect to metagenomic sequencing is obtaining adequate depth and breadth of coverage from pathogens like RNA viruses that i) typically have small genomes, and ii) are typically present at low titers amidst the background ‘noise’ of the host and commensals [1]. Organisms with larger genomes have the potential to contribute a larger proportion to the overall number of sequencing reads even when the plaque-forming units (PFU), or colony-forming units (CFU) in the case of bacteria, are equivalent to that of an organism with a smaller sized genome
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
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 call
