A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses

Abstract

Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.

Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA) Enable High-Throughput and Comprehensive Virus Identification

“…It is also known that index hopping is a common issue when performing multiplex sequencing. We applied 0.1% as the index hopping rate and Poisson process to estimate the possible number of reads caused by the index hopping ( 18 , 19 ). Then, the number of reads (μ) that resulted in an upper cumulative Poisson probability P ( x ≥ μ) of <0.01 was determined as the cutoff value.…”

Peptide-Based Flavivirus Biosensors: From Cell Structure to Virological and Serological Detection Methods

“…In contrast, serological detection is based on the immunological mechanism of antibodies that are released in response to virus infection into the blood serum of the infected person . However, the conventional detection method is time-consuming, complex, and only provides semiquantitative results . Low DNA/RNA concentrations in samples may cause the procedure to fail .…”

“…14 However, the conventional detection method is timeconsuming, complex, and only provides semiquantitative results. 15 Low DNA/RNA concentrations in samples may cause the procedure to fail. 16 Serological detection needs at least 3−5 days to wait for a sufficient number of antibodies to be produced after the onset of flavivirus illness, and there may be cross-reactivity with other flaviviruses during the waiting period.…”