Laboratory of Medical Microbiology, Department of Microbiology, Hellenic Pasteur Institute, 11521 Athens, Greece.
Bioinformatics and Applied Genomics Unit, Department of Microbiology, Hellenic Pasteur Institute, 11521 Athens, Greece.
Viruses. 2022 Jun 11;14(6):1272. doi: 10.3390/v14061272.
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.
靶向病毒组富集和测序（VirCapSeq-VERT）利用寡核苷酸（探针）池来富集所有已知的脊椎动物感染病毒，最高可达 2015 年，从而提高检测灵敏度。探针与目标序列的杂交可以是部分的，从而能够检测和基因组重建与用于探针设计的菌株相比具有<40%遗传多样性的新型病原体。在这项研究中，我们在病毒提取物的多路混合中部署了这种方法，并评估了它在 cDNA 合成后对 DNA 和 RNA 病毒进行无偏检测的性能。我们进一步评估了它在去除各种背景基因组材料方面的效率。最后，作为概念验证，我们探索了该方法在表征未知新兴人类病毒（如 SARS-CoV-2）方面的潜在用途，这些病毒可能不在探针面板中。我们混合了 SARS-CoV-2、冠状病毒 OC43、巨细胞病毒、甲型流感病毒 H3N2、细小病毒 B19、呼吸道合胞病毒、腺病毒 C 和柯萨奇病毒 A16 的等摩尔 DNA/RNA 病毒提取物的阳性样本。对 dsDNA 混合物进行靶向病毒组富集，然后在 NextSeq500（Illumina）和便携式 MinION 测序仪上进行测序，以评估其作为即时护理（PoC）应用的可用性。使用病毒参考序列进行基因组映射组装。未靶向文库中大多数病毒基因组的映射总读数不到 1%，而 RNA 病毒仍未检测到。在靶向文库中，病毒映射读取的百分比大大增加，大多数情况下允许进行全基因组组装。靶向病毒组测序可以富集广泛的病毒，可能能够发现新兴病毒。
