期刊
VIRUSES-BASEL
卷 14, 期 6, 页码 -出版社
MDPI
DOI: 10.3390/v14061272
关键词
target enrichment; virome sequencing; SARS-CoV-2; COVID-19; NGS diagnostics; emerging viruses; nanopore sequencing
类别
资金
- European Union (European Social Fund-ESF) through the Operational Program Human Resources Development, Education and Lifelong Learning 2014-2020 [MIS: 5050220]
Targeted virome enrichment and sequencing (VirCapSeq-VERT) is an effective method for detecting DNA and RNA viruses in viral extracts. It has the ability to detect viruses with low genetic diversity and remove background genomic material. This method can potentially be used to discover emerging viruses.
Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known-up to 2015-vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits' design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits' panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses.
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