Deep sequencing as diagnostic tool for highly pathogenic viruses

Abstract
With the advent of the real-time PCR nucleic acid-based diagnostics has become the gold standard for the identification of viral and bacterial pathogens in clinical as well as in environmental samples. Real-time PCR is fast, reliable, and due to the application of specific “probes” it offers an additional level of specificity. Because of this pronounced specificity, however, PCR-based techniques may often fail to detect new or emerging pathogens with differing or so far unknown genetic information. While multiplex PCR systems are a convenient approach to specifically detect a wider variety of pathogens in one reaction vessel, generic PCR-based amplification systems offer a more open view and can even facilitate the detection of new pathogens.
Compared to electron microscopy, which provides an actual diagnostic open view with serious restrictions regarding the detection limit, recently metagenomic approaches based on massively parallel sequencing techniques have promised to be a more sensitive valuable tool for the detection of unknown pathogens. Since it is technically possible to gain sequence information of all pathogens present in a particular sample, the most challenging task is to identify the sequences of interest in the bulk of sequence data obtained by only one sequencing run. In this presentation the benefits and drawbacks of next generation sequencing as diagnostic tool will be discussed in comparison to conventional methods of virus detection.