Sensitivity and specificity of metatranscriptomics as an arbovirus surveillance tool
journal contributionposted on 07.01.2021, 05:22 by Jana BatovskaJana Batovska, PT Mee, SE Lynch, Tim SawbridgeTim Sawbridge, Brendan RodoniBrendan Rodoni
© 2019, The Author(s). 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.
The authors thank Russell Barnes for collecting the mosquitoes and Karen Brown for morphologically identifying them; Jonathan Darbro and Elise Kho for providing the Ross River virus-infected Aedes notoscriptus specimens; and Tracie Webster for her assistance in sequencing the samples and re-demultiplexing the reads. The authors also thank the two anonymous reviewers whose important feedback helped to improve this manuscript. This work was funded by the Biosciences Research Innovation Fund Program provided by the Victorian Department of Jobs, Precincts and Regions. The mosquito trapping was performed as part of the Victorian Arbovirus Disease Control Program, which is funded by the Department of Health and Human Services. JB is supported by an Australian Government Research Training Program Scholarship.
Biosciences Research Innovation Fund Program by the Victorian Department of Jobs, Precincts and Regions
Department of Health and Human Services
Australian Government Research Training Program Scholarship
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Science & TechnologyMultidisciplinary SciencesScience & Technology - Other TopicsRNA VIRUS METAGENOMICSVIRAL METAGENOMICSMOSQUITOSVIROMEIDENTIFICATIONTRANSCRIPTOMEVALIDATIONPATHOGENSORBIVIRUSDEPLETIONAnimalsArbovirusesAustraliaCulexCulicidaeHumansMetagenomeMosquito VectorsRNA, ViralRoss River virusSensitivity and SpecificityTranscriptome