Assessment of metagenomic Nanopore and Illumina sequencing for recovering whole genome sequences of chikungunya and dengue viruses directly from clinical samples

Abstract

BackgroundThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. AimTo investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples.MethodsWe performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study.ResultsDirect metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV. ConclusionsThis work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.

Keywords: chikungunya; chikungunya virus; dengue; dengue fever; dengue virus; metagenomic; molecular methods; nanopore; surveillance; typing; vector-borne infections; viral infections.

Figure 1

Cycle threshold (Ct) values distribution of chikungunya (n = 73) and dengue virus (n = 368) positive samples from the Rare and Imported Pathogens Laboratory, Public Health England, United Kingdom, 2016 (n = 441 total samples)

Figure 2

Proportion of reads mapping to the appropriate viral reference sequence and proportion of reference genome sequenced at minimum 20-fold coverage in each chikungunya or dengue virus positive sample, United Kingdom, 2017^a (n = 26 samples)

Figure 3

Comparison of Nanopore and Illumina results, as to proportions of reads mapping to the appropriate reference viral sequence, and proportions of reference genome sequenced at minimum 20-fold coverage, United Kingdom, 2017^a (n = 8 samples)

Figure 4

Coverage depth across the chikungunya or dengue viral genome, United Kingdom, 2017^a (n = 8 samples)

Figure 5

Kraken classification of reads from metagenomic sequencing in (A) chikungunya and (B) dengue real-time reverse transcription-PCR positive samples, United Kingdom, 2017^a (n = 8 samples)

Figure 6

Comparison of genome coverage depth across the chikungunya virus or dengue virus genome for different sequencing library preparation methods in a sample coinfected with dengue and chikungunya viruses, United Kingdom, 2017^a (n = 1 sample)