Sequencing needs for viral diagnostics

Abstract

We built a system to guide decisions regarding the amount of genomic sequencing required to develop diagnostic DNA signatures, which are short sequences that are sufficient to uniquely identify a viral species. We used our existing DNA diagnostic signature prediction pipeline, which selects regions of a target species genome that are conserved among strains of the target (for reliability, to prevent false negatives) and unique relative to other species (for specificity, to avoid false positives). We performed simulations, based on existing sequence data, to assess the number of genome sequences of a target species and of close phylogenetic relatives (near neighbors) that are required to predict diagnostic signature regions that are conserved among strains of the target species and unique relative to other bacterial and viral species. For DNA viruses such as variola (smallpox), three target genomes provide sufficient guidance for selecting species-wide signatures. Three near-neighbor genomes are critical for species specificity. In contrast, most RNA viruses require four target genomes and no near-neighbor genomes, since lack of conservation among strains is more limiting than uniqueness. Severe acute respiratory syndrome and Ebola Zaire are exceptional, as additional target genomes currently do not improve predictions, but near-neighbor sequences are urgently needed. Our results also indicate that double-stranded DNA viruses are more conserved among strains than are RNA viruses, since in most cases there was at least one conserved signature candidate for the DNA viruses and zero conserved signature candidates for the RNA viruses.

FIG. 1.

Range plot for plum pox virus, displaying results typical of many viruses. To discriminate samples in which zero NNs were used, the range is drawn as a horizontal grey line, and when n is >0, the range is drawn as a black line. The best estimate of the true value is the quality measure determined using the entire target and NN pools and is represented by a vertical black line. This best estimate plus a constant c of 20 is at the location of the vertical dashed line and was selected to indicate a reasonable distance from the true answer. The 75% quantile for each range is shown with a black, vertical tick mark.

FIG. 2.

Range plot for vaccinia, illustrating that NN sequence information is critical to eliminate signature candidates that are not species specific. Three or four each of target and NN sequences appear to be adequate for prediction of a short list of signature candidates that are suitable for laboratory screening.

FIG. 3.

Range plot for SARS, similar to that for Ebola Zaire (data not shown), illustrates that additional target sequence data do not narrow the list of signature candidates. No NN sequences available at the time of these analyses were similar enough to winnow the list of signature candidates.