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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

Rarefaction analysis showing the covered proportion of the EV genome as a function of the total number of sequence reads from each sample for RNA shotgun sequencing.Numbers provided for each sample are given as copies/μl in the test sample.
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pone.0122636.g003: Rarefaction analysis showing the covered proportion of the EV genome as a function of the total number of sequence reads from each sample for RNA shotgun sequencing.Numbers provided for each sample are given as copies/μl in the test sample.

Mentions: Coverage of the EV, aRNA, HPV-18, and HIV-1 RNA genomes was investigated. The sample with the highest concentration of EV (12,500 copies/μl) yielded sufficient reads to completely cover the EV reference genome. For the remaining samples the genome was partially covered. Results showed that the coverage approached saturation for all samples, and deeper sequencing was unlikely to result in additional unique viral reads at any of the concentrations (Fig 3).


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)

Rarefaction analysis showing the covered proportion of the EV genome as a function of the total number of sequence reads from each sample for RNA shotgun sequencing.Numbers provided for each sample are given as copies/μl in the test sample.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122636.g003: Rarefaction analysis showing the covered proportion of the EV genome as a function of the total number of sequence reads from each sample for RNA shotgun sequencing.Numbers provided for each sample are given as copies/μl in the test sample.
Mentions: Coverage of the EV, aRNA, HPV-18, and HIV-1 RNA genomes was investigated. The sample with the highest concentration of EV (12,500 copies/μl) yielded sufficient reads to completely cover the EV reference genome. For the remaining samples the genome was partially covered. Results showed that the coverage approached saturation for all samples, and deeper sequencing was unlikely to result in additional unique viral reads at any of the concentrations (Fig 3).

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