The use of next generation sequencing in the diagnosis and typing of respiratory infections

Abstract

Background: Molecular assays are the gold standard methods used to diagnose viral respiratory pathogens. Pitfalls associated with this technique include limits to the number of targeted pathogens, the requirement for continuous monitoring to ensure sensitivity/specificity is maintained and the need to evolve to include emerging pathogens. Introducing target independent next generation sequencing (NGS) could resolve these issues and revolutionise respiratory viral diagnostics.

Objectives: To compare the sensitivity and specificity of target independent NGS against the current standard diagnostic test.

Study design: Diagnostic RT-PCR of clinical samples was carried out in parallel with target independent NGS. NGS sequences were analyzed to determine the proportion with viral origin and consensus sequences were used to establish viral genotypes and serotypes where applicable.

Results: 89 nasopharyngeal swabs were tested. A viral pathogen was detected in 43% of samples by NGS and 54% by RT-PCR. All NGS viral detections were confirmed by RT-PCR.

Conclusions: Target independent NGS can detect viral pathogens in clinical samples. Where viruses were detected by RT-PCR alone the Ct value was higher than those detected by both assays, suggesting an NGS detection cut-off - Ct=32. The sensitivity and specificity of NGS compared with RT-PCR was 78% and 80% respectively. This is lower than current diagnostic assays but NGS provided full genome sequences in some cases, allowing determination of viral subtype and serotype. Sequencing technology is improving rapidly and it is likely that within a short period of time sequencing depth will increase in-turn improving test sensitivity.

Keywords: Diagnostics; Next generation sequencing; Viral respiratory infection; Virus detection;.

Fig. 1

HRV genome coverage – a graphic visualization of the reference genome coverage following Bowtie2 alignment of sequencing reads to the top BLAST hit for each HRV positive sample.

Fig. 2

Neighbor-joining (NJ) phylogenetic tree of full-genome reference sequences and a subset of the NGS consensus sequences with full or near-full reference genome coverage. The branch annotations represent the bootstrap values (percentage of 1000 samples trees). Sequences generated in this study are shown as circles and squares.

Fig. 3

Threshold cycle (Ct) values respiratory of samples positive by RT-PCR and either confirmed or unconfirmed by the NGS assay. The bars indicate the mean and one standard deviation. An unpaired t-test demonstrates a significant difference between the two groups (p < 0.0001).

Fig. 4

The percentage of sequenced reads, after quality trimming, mapping to the taxonomic reference genome determined by BLAST. Linear regression of data, R² value = 0.19 (p = 0.0057). This is suggestive of a log relationship between the proportion of reads mapping and the Ct value.