Evaluation of High-Throughput Sequencing for Identifying Known and Unknown Viruses in Biological Samples

Abstract

High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and has rapidly become a major tool for identifying viruses in biological samples, and in particular when the target sequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of viruses in biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms. We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed of single or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were added at a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection of quantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented, in public nucleotide sequence databases, we show that the higher output of Illumina is associated with a much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study, identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations, the number of reads generated by the Illumina platform was too small to facilitate assembly of contigs without the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load was sufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-length genomes and thus should facilitate the identification of novel viruses.

Identical Consensus Sequence and Conserved Genomic Polymorphism of Hepatitis E Virus during Controlled Interspecies Transmission

“…After DNase treatment for 2 h at 37°C (0.33 U/l of sample; Qiagen, France), total nucleic acids were extracted using a Nucleospin RNA virus kit (Macherey-Nagel, Germany) and then amplified without use of HEV-specific PCR primers, as described previously (6). Briefly, bacteriophage ⌽29 polymerase-based multiple-displacement amplification was preceded by a cDNA synthesis step performed with random hexamer primers.…”

Landscape features and helminth co-infection shape bank vole immunoheterogeneity, with consequences for Puumala virus epidemiology

“…More detailed investigations of the whole pathogen communities infecting these bank voles are now possible from the development of new generation sequencing technologies and metagenomic analyses. These approaches have yet allowed to describe microbial communities and to explore their interactions in different hosts' organs or environments (Nakamura et al 2008;Carpi et al, 2011;Cheval et al, 2011). In the future, the integration of such information in the analysis of PUUV epidemiology will provide a better understanding of the impacts of co-infection on the evolution of tolerance to PUUV in bank voles.…”

Next-generation sequencing and bioinformatic approaches to detect and analyze influenza virus in ferrets

“…The selection of the sequencing platform will determine the number of reads that can be obtained. Roche 454 FLX GS was the first NGS platform commercially available; however, its technical capacities were surpassed shortly after by the Illumina sequencers [30]. We sequenced one RNA sample using both platforms, and although the experimental design was not intended to make direct comparisons between technologies, we were able to conclude that direct RNA sequencing in the 454 platform can resolve only the most highly expressed viral genes (as shown in results), a scenario that highly resembles the results from a previously published paper [7]; therefore, viral analysis by 454 sequencing requires prior enrichment of the viral segments by PCR amplification or sequence-specific capture.…”