دورية أكاديمية
Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing [version 2; peer review: 2 approved]
| العنوان: | Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing [version 2; peer review: 2 approved] |
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| المؤلفون: | Claro, IM, Ramundo, MS, Coletti, TM, Da Silva, CAM, Valenca, IN, Candido, DS, Sales, FCS, Manuli, ER, De Jesus, JG, De Paula, A, Felix, AC, Andrade, PDS, Pinho, MC, Souza, WM, Amorim, MR, Proenca-Modena, JL, Kallas, EG, Levi, JE, Faria, NR, Sabino, EC, Loman, NJ, Quick, J |
| بيانات النشر: | Wellcome |
| سنة النشر: | 2023 |
| المجموعة: | Imperial College London: Spiral |
| جغرافية الموضوع: | England |
| الوصف: | Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| تدمد: | 2398-502X |
| العلاقة: | Wellcome Open Research; http://hdl.handle.net/10044/1/107278Test |
| DOI: | 10.12688/wellcomeopenres.17170.2 |
| الإتاحة: | https://doi.org/10.12688/wellcomeopenres.17170.2Test http://hdl.handle.net/10044/1/107278Test |
| حقوق: | © 2023 Claro IM et al. This is an open access work distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ; https://creativecommons.org/licenses/by/4.0Test/ |
| رقم الانضمام: | edsbas.5931BAE9 |
| قاعدة البيانات: | BASE |
Full Text Finder | تدمد: | 2398502X |
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| DOI: | 10.12688/wellcomeopenres.17170.2 |