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.

Clinical and biological insights from viral genome sequencing

“…Non-specific amplification methods, such as multiple displacement amplification (MDA), which make use of random primers and Φ29 polymerases, can increase the DNA yield. However, these approaches are time consuming, costly, and may increase the risk of biases, errors and contamination, without necessarily improving sensitivity 78,79 . Moreover, the proportion of host reads often remains high 80 .…”

Promises and pitfalls of using high‐throughput sequencing for diet analysis

“…This measure reduces the probability of type I errors, thus may be desirable in studies where avoiding false positives is essential. However, removing all sequences that appear in negative controls might elevate the rate of type II errors (false negatives), since the negative controls are more prone to contamination by DNA present in abundant quantities and in many samples (Jensen et al, 2015). Therefore, before deciding on how to incorporate controls in the study, for example, whether to use the same negative controls in all steps or use unique controls for each step, it is essential to carefully think about how to use the controls in the bioinformatic processing of the sequences and the consequences in relation to the research question.…”

Current Trends in Diagnostics of Viral Infections of Unknown Etiology

“…Additionally, listed techniques are expensive and laborious. Unspecific amplification methods, e.g., multiple displacement amplification (MDA), utilize random primers and Phi29 polymerase to increase reaction output, however not necessarily with high enrichment quality [104], and may lead to additional contamination, uneven fragment distribution and amplification errors, lowering the overall library quality [105]. Sometimes depletion protocols do not effectively remove host-cell DNA [106].…”

“…Target probe-based enrichment (also known as hybridization-based enrichment) is used for studying viral genomes without prior cultivation or clonal amplification [105][106][107][108][109][110][111][112][113][114][115][116][117]. For this, short DNA or RNA probes are used, which are complementary to the chosen sequences in a viral genome.…”