Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

Barzon, Luisa; Lavezzo, Enrico; Militello, Valentina; Toppo, Stefano; Palù, Giorgio

Like viral metagenomics, sequencing of full-length viral genomes is a difficult task due to the presence of contaminating nucleic acids of the host cell and other agents in viral isolates. In fact, preparation of a simple shotgun sequencing DNA library, the most comprehensive approach, or of a library of cDNA synthesized from RNA with random priming, results in a huge amount of host specific instead of a comprehensive representation of the viral sequences, even in the presence of a very high viral load [21, 31, 73] . Very high throughput sequencing techniques, such as SOLiD platform, could be used to obtain sufficient sequence coverage [74] , but the length of reads might be too short to allow de novo assembly of viral genomes and methods that provide longer reads, like 454 and Illumina technology, might be preferable [31, 32] . Several techniques have been used to enrich virions or viral nucleic acids from cell culture or from host tissue and fluids before extracting the genomic DNA/RNA, in order to limit the contamination from host nucleic acids. One of these methods is ultracentrifugation, but this procedure may be very time-consuming and laborious with uncertain outcome [75] . Other methods are based on enrichment of viral nucleic acids by using capture probes or PCR amplification targeting conserved genome segments [76, 77] or, vice versa, by depletion of host nucleic acids by probing total RNA with labeled host nucleic acid [78] . Other approaches could be enrichment of dsRNA virus genomes [79] or circular dsDNA viral genomes by RCA [28, 29] .