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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: 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.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.

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 (A) HAdV genome, (B) the HPV-18 genome, (C) the HI-1 genome, and (D) the MeV plasmid, as a function of the total number of sequence reads from each sample for DNA shotgun sequencing.Numbers provided for each sample are given as copies/μl in the test sample.
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pone.0122636.g002: Rarefaction analysis showing the covered proportion of the (A) HAdV genome, (B) the HPV-18 genome, (C) the HI-1 genome, and (D) the MeV plasmid, as a function of the total number of sequence reads from each sample for DNA shotgun sequencing.Numbers provided for each sample are given as copies/μl in the test sample.

Mentions: The two samples with the highest concentrations of HAdV (2,500 and 250 copies/μl, respectively), yielded sufficient reads to completely cover the HAdV reference genome. For the lower concentrations, the genome was only partially covered. Rarefaction analysis was employed for analysing species richness by assessing the number of new sequences found in a set interval of sequences. Results showed that the coverage approached saturation in the rarefaction analysis, hence deeper sequencing would predictably result in little additional unique viral reads (Fig 2A).


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 (A) HAdV genome, (B) the HPV-18 genome, (C) the HI-1 genome, and (D) the MeV plasmid, as a function of the total number of sequence reads from each sample for DNA 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.g002: Rarefaction analysis showing the covered proportion of the (A) HAdV genome, (B) the HPV-18 genome, (C) the HI-1 genome, and (D) the MeV plasmid, as a function of the total number of sequence reads from each sample for DNA shotgun sequencing.Numbers provided for each sample are given as copies/μl in the test sample.
Mentions: The two samples with the highest concentrations of HAdV (2,500 and 250 copies/μl, respectively), yielded sufficient reads to completely cover the HAdV reference genome. For the lower concentrations, the genome was only partially covered. Rarefaction analysis was employed for analysing species richness by assessing the number of new sequences found in a set interval of sequences. Results showed that the coverage approached saturation in the rarefaction analysis, hence deeper sequencing would predictably result in little additional unique viral reads (Fig 2A).

Bottom Line: 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.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.

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