A Versatile Processing Workflow to Enable Pathogen Detection in Clinical Samples from Organs Using VIDISCA

Abstract

In recent years, refined molecular methods coupled with powerful high throughput sequencing technologies have increased the potential of virus discovery in clinical samples. However, host genetic material remains a complicating factor that interferes with discovery of novel viruses in solid tissue samples as the relative abundance of the virus material is low. Physical enrichment processing methods, although usually complicated, labor-intensive, and costly, have proven to be successful for improving sensitivity of virus detection in complex samples. In order to further increase detectability, we studied the application of fast and simple high-throughput virus enrichment methods on tissue homogenates. Probe sonication in high EDTA concentrations, organic extraction with Vertrel™ XF, or a combination of both, were applied prior to chromatography-like enrichment using Capto™ Core 700 resin, after which effects on virus detection sensitivity by the VIDISCA method were determined. Sonication in the presence of high concentrations of EDTA showed the best performance with an increased proportion of viral reads, up to 9.4 times, yet minimal effect on the host background signal. When this sonication procedure in high EDTA concentrations was followed by organic extraction with Vertrel™ XF and two rounds of core bead chromatography enrichment, an increase up to 10.5 times in the proportion of viral reads in the processed samples was achieved, with reduction of host background sequencing. We present a simple and semi-high-throughput method that can be used to enrich homogenized tissue samples for viral reads.

Keywords: VIDISCA-NGS; atypical porcine pestivirus; enrichment; metagenomics; virus discovery.

Figure 1

Effect of the novel semi-high-throughput processing workflows on the (A) percentage of viral reads and (B) total number of reads after each Capto™ Core 700 chromatography round.