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.

Fig. 1.

Distribution of the coverage resulting from de novo assembling or mapping on reference sequences of Roche-454 and Illumina reads. (A) Coverage of the anellovirus sequence by the contigs resulting from de novo assembling of Roche-454 and Illumina reads. Depth (y axis) corresponds to the number of reads representing a given nucleotide in the reconstructed sequence. The x axis corresponds to nucleotide positions in the whole genome. One contig from the Roche-454 covered 93.7% of the genome. Two contigs from the Illumina reads covered 92.6% of the genome. (B) Resequencing coverage of the genome of CVS strain of RABV from the hits directly mapped on the reference sequence. Note the difference of scale for the depth between the three graphs.