Abstract
The ability to identify all the viruses within a sample makes metatranscriptomic sequencing an attractive tool to screen mosquitoes for arboviruses. Practical application of this technique, however, requires a clear understanding of its analytical sensitivity and specificity. To assess this, five dilutions (1:1, 1:20, 1:400, 1:8,000 and 1:160,000) of Ross River virus (RRV) and Umatilla virus (UMAV) isolates were spiked into subsamples of a pool of 100 Culex australicus mosquitoes. The 1:1 dilution represented the viral load of one RRV-infected mosquito in a pool of 100 mosquitoes. The subsamples underwent nucleic acid extraction, mosquito-specific ribosomal RNA depletion, and Illumina HiSeq sequencing. The viral load of the subsamples was also measured using reverse transcription droplet digital PCR (RT-ddPCR) and quantitative PCR (RT-qPCR). Metatranscriptomic sequencing detected both RRV and UMAV in the 1:1, 1:20 and 1:400 subsamples. A high specificity was achieved, with 100% of RRV and 99.6% of UMAV assembled contigs correctly identified. Metatranscriptomic sequencing was not as sensitive as RT-qPCR or RT-ddPCR; however, it recovered whole genome information and detected 19 other viruses, including four first detections for Australia. These findings will assist arbovirus surveillance programs in utilising metatranscriptomics in routine surveillance activities to enhance arbovirus detection.
Highlights
The ability to identify all the viruses within a sample makes metatranscriptomic sequencing an attractive tool to screen mosquitoes for arboviruses
Index cross-talk occurred during the sequencing run, with unindexed PhiX reads detectable in every sample
Detection of the spiked viruses using metatranscriptomic sequencing was first evaluated based on the percent genome coverage of the spiked virus by assembled contigs (Percent Coverage by Contigs - PCC) (Table 1)
Summary
The ability to identify all the viruses within a sample makes metatranscriptomic sequencing an attractive tool to screen mosquitoes for arboviruses. Metatranscriptomics is a nontargeted method that offers many advantages for arbovirus surveillance programs It can detect viruses without culturing them, does not require a priori knowledge of the viral sequence, has the potential to identify new arboviral threats, elucidates mixed infections, and can provide whole genome or specific protein sequences for molecular epidemiological investigations of outbreaks[9]. It can detect other organisms in a mosquito pool, including endosymbionts such as Wolbachia[10], and parasites such as Leishmania[11]. A variety of rRNA depletion kits are available, these are not specific to mosquitoes and so custom probes based on mosquito rRNA sequences need to be generated[20,21]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
