Evolution of selective-sequencing approaches for virus discovery and virome analysis

Abstract

Recent advances in sequencing technologies have transformed the field of virus discovery and virome analysis. Once mostly confined to the traditional Sanger sequencing based individual virus discovery, is now entirely replaced by high throughput sequencing (HTS) based virus metagenomics that can be used to characterize the nature and composition of entire viromes. To better harness the potential of HTS for the study of viromes, sample preparation methodologies use different approaches to exclude amplification of non-viral components that can overshadow low-titer viruses. These virus-sequence enrichment approaches mostly focus on the sample preparation methods, like enzymatic digestion of non-viral nucleic acids and size exclusion of non-viral constituents by column filtration, ultrafiltration or density gradient centrifugation. However, recently a new approach of virus-sequence enrichment called virome-capture sequencing, focused on the amplification or HTS library preparation stage, was developed to increase the ability of virome characterization. This new approach has the potential to further transform the field of virus discovery and virome analysis, but its technical complexity and sequence-dependence warrants further improvements. In this review we discuss the different methods, their applications and evolution, for selective sequencing based virome analysis and also propose refinements needed to harness the full potential of HTS for virome analysis.

Fig. 1

Experimental outline of virus enrichment and selective sequencing approaches. (A). Details of the pre-extraction virus enrichment based selective sequencing. Briefly, samples are filtered and/or centrifuged before treatment with nucleases (DNAse, RNAse, Benzonase etc.), followed by their amplification and sequencing. (B). Outline of negative (non-viral probes) and positive (viral probes) selection method using hybridization based capture and selective enrichment of virus nucleic acids, followed by amplification and sequencing. Sequencing data analysis requires bioinformatics based virus discovery or virome analysis and experimental validation of results.