Multiple approaches for massively parallel sequencing of HCoV-19 (SARS-CoV-2) genomes directly from clinical samples

Xiao, Minfeng; Liu, Xiaoqing; Ji, Jingkai; Li, Min; Li, Jiandong; Yang, Lin; Sun, Wanying; Ren, Peng; Yang, Guifang; Zhao, Jincun; Liang, Tianzhu; Ren, Huahui; Chen, Tian; Huang, Zhong; Song, Wenchen; Wang, Yanqun; Deng, Ziqing; Zhao, Yongqiang; Ou, Zhihua; Wang, Daxi; Cai, Jielun; Cheng, Xinyi; Feng, Taiqing; Wu, Honglong; Gong, Yanping; Yang, Huanming; Wang, Jian; Xu, Xun; Zhu, Shida; Chen, Fang; Zhang, Yanyan; Chen, Weijun; Li, Yimin; Li, Junhua

doi:10.1101/2020.03.16.993584

Cited by 16 publications

(24 citation statements)

References 43 publications

(44 reference statements)

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“…Human DNA was removed using DNase I and RNA concentration was measured using Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). After human DNA-depletion, the samples were RNA purified and then subjected to double-stranded DNA library construction using the MGIEasy RNA Library preparation reagent set (MGI, Shenzhen, China) following the method used in the previous study [17]. Possible contamination during experimental processing was tracked using human breast cell lines (Michigan Cancer Foundation-7).…”

Section: Real-time Rt-qpcr and Sequencingmentioning

confidence: 99%

“…The constructed libraries were converted to DNA nanoballs (DNBs) and then sequenced on the DNBSEQ-T7 platform (MGI, Shenzhen, China), generating paired-end short reads with 100bp in length. Most samples were also sequenced using hybrid capture-based enrichment approach that was described in previous study [17]. Briefly, the SARS-…”

Section: Real-time Rt-qpcr and Sequencingmentioning

confidence: 99%

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Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2

Wang¹,

Wang²,

Sun³

et al. 2020

Preprint

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The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions.Author summaryIn this study, we compared SARS-CoV-2 populations of 13 Chinese COVID-19 patients. Those viral populations contained a considerable proportion of viral sub-genomic messenger RNAs (sgmRNA), reflecting an active viral replication activity in the respiratory tract tissues. The comparison of 66 identified intra-host variants further showed a low viral genetic distance between intra-household patients and a narrow transmission bottleneck size. Despite the co-existence of genetically distinct viruses within the same host, most intra-host minor variants were not shared between transmission pairs, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Furthermore, the narrow bottleneck and active viral activity in the respiratory tract show that the passage of a small number of virions can cause infection. Our data have therefore delivered a key genomic resource for the SARS-CoV-2 transmission research and enhanced our understanding of the evolutionary dynamics of SARS-CoV-2.

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Section: Real-time Rt-qpcr and Sequencingmentioning

confidence: 99%

Section: Real-time Rt-qpcr and Sequencingmentioning

confidence: 99%

Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2

Wang¹,

Wang²,

Sun³

et al. 2020

Preprint

Self Cite

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“…The application of next-generation sequencing can be an accurate diagnosis method for SARS-CoV-2, including metagenomics, hybrid capture-based and amplicon-based next-generation sequencing 1, 4, 5 . These three approaches show a higher sensitivity than conventional RT-PCR, and can meet the needs of secondary detection, double-check diagnosis and large-scale suspected sample detection of RT-PCR false-negative samples5 . However, the high cost is the most important obstacle to popularize virus sequencing nowadays.…”

mentioning

confidence: 99%

Diagnostic options for coronavirus disease 2019 (COVID-19)

Xiao

Peng

Tan

et al. 2020

Infect. Control Hosp. Epidemiol.

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“…Target enrichment with spiked-in primers can improve SARS-CoV-2 genome coverage( 5 ), but the reliance on specific primers inherently limits this approach for the profiling of fast evolving viruses such as coronaviruses. The same limitation applies to multiplex RT-PCR-based strategies( 6 ). Additionally, once the sample is subject to targeted amplification during the initial reverse transcription (RT) steps, its metatranscriptomic information is lost forever.…”

Section: Introductionmentioning

confidence: 99%

“…Currently, the most comprehensive strategy is the combination of metatranscriptomics profiling with post-library SARS-CoV-2 target enrichment( 6 ). However, in most conventional RNA-seq methods, the double-strand DNA ligation (dsDL) portion of the protocol is usually the most demanding on hands-on time and user technique( 7 ).…”

Section: Introductionmentioning

confidence: 99%

MINERVA: A facile strategy for SARS-CoV-2 whole genome deep sequencing of clinical samples

Chen¹,

Lin

et al. 2020

Preprint

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32The novel coronavirus disease 2019 pandemic poses a serious public health 33 risk. Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-34 CoV-2) from clinical samples is crucial for the understanding of viral spread and viral 35 evolution, as well as for vaccine development. Existing sample preparation methods for 36 viral genome sequencing are demanding on user technique and time, and thus not ideal 37 for time-sensitive clinical samples; these methods are also not optimized for high 38 performance on viral genomes. We have developed MetagenomIc RNA EnRichment 39 VirAl sequencing (MINERVA), a facile, practical, and robust approach for metagenomic 40 and deep viral sequencing from clinical samples. This approach uses direct tagmentation 41 of RNA/DNA hybrids using Tn5 transposase to greatly simplify the sequencing library 42 construction process, while subsequent targeted enrichment can generate viral genomes 43 with high sensitivity, coverage, and depth. We demonstrate the utility of MINERVA on 44 pharyngeal, sputum and stool samples collected from COVID-19 patients, successfully 45 obtaining both whole metatranscriptomes and complete high-depth high-coverage SARS-46 CoV-2 genomes from these clinical samples, with high yield and robustness. MINERVA 47 is compatible with clinical nucleic extracts containing carrier RNA. With a shortened 48 hands-on time from sample to virus-enriched sequencing-ready library, this rapid, 49 versatile, and clinic-friendly approach will facilitate monitoring of viral genetic variations 50 during outbreaks, both current and future. 51 52 53 Introduction 54As of April 24, 2020, the ongoing COVID-19 viral pandemic has affected more than 2.6 55 million people in over 200 countries and territories around the world, and has claimed 56 more than 180 thousand lives. Closely monitoring the genetic diversity and distribution of 57 viral strains at the population level is essential for epidemiological tracking, and for 58 understanding viral evolution and transmission; additionally examining the viral 59 heterogeneity within a single individual is imperative for diagnosis and treatment. The 60 disease-causing pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-61 CoV-2), was identified from early disease cases and its draft genome sequenced within 62 weeks, thanks to the rapid responses from researchers around the world(1, 2). The initial 63 SARS-CoV-2 draft genome was obtained independently from the same early COVID-19 64 patient samples using various conventional RNA-seq sequencing library construction 65 methods. Although these library construction methods successfully generated a draft 66 genome, several drawbacks hinder the use of these methods for routine viral genome 67 sequencing from the surge of clinical samples during an outbreak. 68 69 One direct library construction approach which was used to generate the SARS-CoV-2 70 draft genome(1, 2) essentially captures each sample's entire metatranscriptome, in which 71 SARS-CoV-2 is just one species ...

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Multiple approaches for massively parallel sequencing of HCoV-19 (SARS-CoV-2) genomes directly from clinical samples

Cited by 16 publications

References 43 publications

Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2

Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2

Diagnostic options for coronavirus disease 2019 (COVID-19)

MINERVA: A facile strategy for SARS-CoV-2 whole genome deep sequencing of clinical samples

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