title: Target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery.
abstract: Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.
authors: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors: Randi Holm
authors: RH
authors: Jensen
authors: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors: Sarah
authors: S
authors: Mollerup
authors: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors: Tobias
authors: T
authors: Mourier
authors: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors: Thomas Arn
authors: TA
authors: Hansen
authors: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors: Helena
authors: H
authors: Fridholm
authors: Department of Epidemiology Research, Statens Serum Institut, Copenhagen, Denmark.
authors: Lars Peter
authors: LP
authors: Nielsen
authors: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors: Eske
authors: E
authors: Willerslev
authors: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors: Anders Johannes
authors: AJ
authors: Hansen
authors: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors: Lasse
authors: L
authors: Vinner
mesh_terms: D001483:Base Sequence; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D052161:Human papillomavirus 18 / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D006801:Humans; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D014771:Virion / Q000235:genetics* / Q000302:isolation & purification; D014777:Virus Diseases / Q000235:genetics* / Q000821:virology
chemical_list: D012367:RNA, Viral