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Target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery.

Jensen RH, Mollerup S, Mourier T, Hansen TA, Fridholm H, Nielsen LP, Willerslev E, Hansen AJ, Vinner L - PLoS ONE (2015)

Bottom Line: However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus

Overview of the experimental design.Fractions of each sample, consisting of varying viral material spiked into human PBMCs were subjected to shotgun sequencing of DNA or RNA. On other fractions virion enrichment procedures including centrifugation, filtration and nuclease treatment were performed prior to library preparation (DNA or RNA) and sequencing.
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pone.0122636.g001: Overview of the experimental design.Fractions of each sample, consisting of varying viral material spiked into human PBMCs were subjected to shotgun sequencing of DNA or RNA. On other fractions virion enrichment procedures including centrifugation, filtration and nuclease treatment were performed prior to library preparation (DNA or RNA) and sequencing.

Mentions: This study aimed at determining the sensitivity of different HTS library preparation procedures commonly used for viral discovery. For decreasing concentrations of target, we compared the effect of virion enrichment. To mimic the complexity of a biological sample, test sample material was generated containing different types of virions and/or infected human cells, spiked to a pool of human peripheral blood mononuclear cells (PBMCs). To represent some of the diversity between viral families, we included viruses with either DNA or RNA genomes, non-enveloped viruses, proviruses integrated into the human genome in single or multiple copies per genome, plasmid DNA, as well as armored RNA (aRNA). An important aspect of this investigation was to determine the level of enrichment compared to the need of sequencing depth in shotgun sequencing. Each sample was split in four fractions of which two were used for shotgun sequencing of total DNA or RNA. The remaining two fractions were subjected to virion enrichment, and libraries were prepared on virion-associated DNA or RNA, from here on referred to as virion-enriched libraries (Fig 1).


Target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery.

Jensen RH, Mollerup S, Mourier T, Hansen TA, Fridholm H, Nielsen LP, Willerslev E, Hansen AJ, Vinner L - PLoS ONE (2015)

Overview of the experimental design.Fractions of each sample, consisting of varying viral material spiked into human PBMCs were subjected to shotgun sequencing of DNA or RNA. On other fractions virion enrichment procedures including centrifugation, filtration and nuclease treatment were performed prior to library preparation (DNA or RNA) and sequencing.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390369&req=5

pone.0122636.g001: Overview of the experimental design.Fractions of each sample, consisting of varying viral material spiked into human PBMCs were subjected to shotgun sequencing of DNA or RNA. On other fractions virion enrichment procedures including centrifugation, filtration and nuclease treatment were performed prior to library preparation (DNA or RNA) and sequencing.
Mentions: This study aimed at determining the sensitivity of different HTS library preparation procedures commonly used for viral discovery. For decreasing concentrations of target, we compared the effect of virion enrichment. To mimic the complexity of a biological sample, test sample material was generated containing different types of virions and/or infected human cells, spiked to a pool of human peripheral blood mononuclear cells (PBMCs). To represent some of the diversity between viral families, we included viruses with either DNA or RNA genomes, non-enveloped viruses, proviruses integrated into the human genome in single or multiple copies per genome, plasmid DNA, as well as armored RNA (aRNA). An important aspect of this investigation was to determine the level of enrichment compared to the need of sequencing depth in shotgun sequencing. Each sample was split in four fractions of which two were used for shotgun sequencing of total DNA or RNA. The remaining two fractions were subjected to virion enrichment, and libraries were prepared on virion-associated DNA or RNA, from here on referred to as virion-enriched libraries (Fig 1).

Bottom Line: However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus