Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery

Abstract

The discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. The preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. Physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. With the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. A review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. These were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as DNA (adenovirus) and RNA viruses (influenza A and human enterovirus), being either non-enveloped capsid or enveloped viruses. The effect of the enrichment method was assessed by both quantitative real-time PCR and metagenomic analysis that incorporated an amplification step. Reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qPCR, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. A 3-step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. This study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. This study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated.

A Pan-HIV Strategy for Complete Genome Sequencing

“…Cultured virus isolates were used as a model system to demonstrate the HIV-SMART principle and the breadth of diversity it could accommodate. A preponderance of metagenomics papers describing new methods have also relied on cultured isolates or transfected plasmids with lower host backgrounds and higher virus titers (13,(29)(30)(31)(32)(33). However, sequencing directly from plasma is the most expedient workflow and reveals which replication-competent viruses are circulating in the patient.…”

“…We optimized the protocol for this purpose and found that the addition of benzonase prior to extraction increased HIV-1 reads (ϳ10-to 50-fold) and genome coverage (from 28% to 54%), presumably by decreasing human background nucleic acid, while a postextraction DNase treatment was often detrimental ( Fig. 3; see Table S2 in the supplemental material) (24,30). The selection of nucleic acid extraction method also drove performance, with higher HIV-1 RNA recovery correlating with higher genome coverage.…”

Virome Analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis Ticks Reveals Novel Highly Divergent Vertebrate and Invertebrate Viruses

“…Ticks were pooled prior to nucleic acid extraction; two pools of I. scapularis (n ϭ 30/pool), two pools of D. variabilis (n ϭ 30/pool), and a single pool of A. americanum (n ϭ 25) were prepared. A two-step purification and nuclease treatment protocol was followed prior to extraction to enrich for viral sequences and minimize host, bacterial, and fungal templates that could compete with the virus template in HTS (22). Each tick pool was homogenized in 500 l of phosphate-buffered saline (PBS), followed by purification through a 0.45-m filter.…”

A major outbreak of conjunctivitis caused by coxsackievirus A24, Réunion, January to April 2015

“…Viral nucleic acid enrichment was performed by a combination of centrifugation (10 min at 6,000 x g), filtration (at 0.45 μm) and nuclease treatment using 0.1 U μL − 1 Turbo DNAse (Life Technologies) as described before [16]. Nucleic acid extraction was then performed with a NucliSens easyMAG magnetic bead system (BioMérieux).…”