Evaluation on the use of Nanopore sequencing for direct characterization of coronaviruses from respiratory specimens, an
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SHORT REPORT
Evaluation on the use of Nanopore sequencing for direct characterization of coronaviruses from respiratory specimens, and a study on emerging missense mutations in partial RdRP gene of SARS‑CoV‑2 Wai Sing Chan1, Chun Hang Au1, Ho Yin Lam1, Candy Ling Na Wang1, Dona Ngar‑Yin Ho1, Yuk Man Lam1, Daniel Ka Wing Chu2, Leo Lit Man Poon2, Tsun Leung Chan1, Jonpaul Sze‑Tsing Zee1, Edmond Shiu Kwan Ma1 and Bone Siu Fai Tang1*
Abstract Coronavirus disease 2019 (COVID-19) pandemic has been a catastrophic burden to global healthcare systems. The fast spread of the etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the need to identify unknown coronaviruses rapidly for prompt clinical and public health decision making. Moreover, owing to the high mutation rate of RNA viruses, periodic surveillance on emerging variants of key virus components is essential for evaluating the efficacy of antiviral drugs, diagnostic assays and vaccines. These 2 knowledge gaps formed the basis of this study. In the first place, we evaluated the feasibility of characterizing coronaviruses directly from respiratory specimens. We amplified partial RdRP gene, a stable genetic marker of coronaviruses, from a collection of 57 clinical specimens positive for SARS-CoV-2 or other human coronaviruses, and sequenced the amplicons with Nanopore Flongle and MinION, the fastest and the most scalable massively-parallel sequencing platforms to-date. Partial RdRP sequences were successfully amplified and sequenced from 82.46% (47/57) of specimens, ranging from 75 to 100% by virus type, with consensus accuracy of 100% compared with Sanger sequences available (n = 40). In the second part, we further compared 19 SARS-CoV-2 RdRP sequences collected from the first to third waves of COVID-19 out‑ break in Hong Kong with 22,173 genomes from GISAID EpiCoV™ database. No single nucleotide variants (SNVs) were found in our sequences, and 125 SNVs were observed from global data, with 56.8% being low-frequency (n = 1–47) missense mutations affecting the rear part of RNA polymerase. Among the 9 SNVs found on 4 conserved domains, the frequency of 15438G > T was highest (n = 34) and was predominantly found in Europe. Our data provided a glimpse into the sequence diversity of a primary antiviral drug and diagnostic target. Further studies are warranted to investi‑ gate the significance of these mutations. Keywords: Coronavirus, COVID-19, Flongle, MinION, Missense mutation, Nanopore, RdRP, SARS-CoV-2
*Correspondence: [email protected] 1 Department of Pathology, Hong Kong Sanatorium and Hospital, Hong Kong, China Full list of author information is available at the end of the article
Background At the time of writing, coronavirus disease 2019 (COVID19) has affected 216 countries, areas or territories, with 9,843,073 confirmed cases and 495,760 confirmed deaths in 6 months from the outbreak in Wuhan, China [1]. In
© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 Inter
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