nCoV-2019 sequencing protocol v1

Quick, Josh

doi:10.17504/protocols.io.bbmuik6w

Cited by 340 publications

(317 citation statements)

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“…In this study, we started using the ARTIC method [14] to amplify the SARS-CoV-2 using a tiling PCR approach. This method recommends the use the TruSeq DNA Nano kit for Illumina sequencing, which is a very labour intensive approach (up to 12h hands on time).…”

Section: Discussionmentioning

confidence: 99%

“…However, our flow cells were over two years old and had less than 600 active pores each, which produced low quality and lower coverage genome (data not shown). We then moved to use the ARTIC protocol [14] on the Illumina Miseq sequencing platform on 8 April 2020.…”

Section: Sars-cov-2 Sequencing With Limited Reagents and Stockout Durmentioning

confidence: 99%

“…We performed cDNA synthesis using SuperScript IV reverse transcriptase (Life Technologies) and random hexamer primers, followed by gene specific multiplex PCR using the ARTIC protocol, as described previously [14]. In Technologies).…”

Section: Tiling-based Polymerase Chain Reactionmentioning

confidence: 99%

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Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation During a Pandemic

Pillay

Giandhari

Tegally

et al. 2020

Preprint

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The COVID-19 pandemic spread very fast around the world. A few days after the first detected case in South Africa, an infection started a large hospital outbreak in Durban, KwaZulu-Natal. Phylogenetic analysis of SARS-CoV-2 genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. In this manuscript, we outline the obstacles we encountered in order to genotype SARS-CoV-2 in real-time during an urgent outbreak investigation. In this process, we encountered problems with the length of the original genotyping protocol, reagent stockout and sample degradation and storage. However, we managed to set up three different library preparation methods for sequencing in Illumina. We also managed to decrease the hands on library preparation time from twelve to three hours, which allowed us to complete the outbreak investigation in just a few weeks. We also fine-tuned a simple bioinformatics workflow for the assembly of high-quality genomes in real-time. In order to allow other laboratories to learn from our experience, we released all of the library preparation and bioinformatics protocols publicly and distributed them to other laboratories of the South African Network for Genomics Surveillance (SANGS) consortium.

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Section: Discussionmentioning

confidence: 99%

Section: Sars-cov-2 Sequencing With Limited Reagents and Stockout Durmentioning

confidence: 99%

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Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation During a Pandemic

Pillay

Giandhari

Tegally

et al. 2020

Preprint

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“…SARS-CoV-2 positive samples were processed for NGS using a highly multiplexed PCR amplicon approach for sequencing on the Oxford Nanopore Technologies (ONT; Oxford, United Kingdom) MinION using the V1 primer pools (Quick et al, 2017) . Sequencing libraries were barcoded and multiplexed using the Ligation Sequencing Kit and Native Barcoding Expansion pack (ONT) following the ARTIC Network's library preparation protocol (Quick, 2020) with the following minor modifications: cDNA was generated with SuperScriptIV VILO Master Mix (ThermoFisher Scientific, Waltham, MA, USA), a total of 20 ng of each sample was used as input into end repair, end repair incubation time was increased to 25 minutes followed by a 1:1 bead-based clean up, and Blunt/TA ligase (New England Biolabs, Ipswich, MA, USA) was used to ligate barcodes to each sample. cDNA synthesis and amplicon generation was performed concurrently for each sample.…”

Section: Sample Collection Processing and Sequencingmentioning

confidence: 99%

Coast-to-coast spread of SARS-CoV-2 in the United States revealed by genomic epidemiology

Fauver

Petrone

Hodcroft

et al. 2020

Preprint

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Since its emergence and detection in Wuhan, China in late 2019, the novel coronavirus SARS-CoV-2 has spread to nearly every country around the world, resulting in hundreds of thousands of infections to date. The virus was first detected in the Pacific Northwest region of the United States in January, 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the U.S., we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated impacts of federal travel restrictions. This study provides evidence for widespread, sustained transmission of SARS-CoV-2 within the U.S. and highlights the critical need for local surveillance.

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“…described in protocol published by ARTIC Network(Quick, 2020) but scaled downed to 1/4.The Ct values in clinical qPCR test for those samples ranged from 25 to 30. The cDNA was diluted to 10-fold by H2O, and 1 μl of the diluted cDNA was used for 10 μl reaction Q5 Hot START DNA Polymerase (NEB) (2 μl of 5x buffer, 0.8 μl of 2.5 mM dNTPs, 0.1 μl of polymerase and 0.29 μl of 50 μM primer mix volumed-up by milli-Q water).…”

mentioning

confidence: 99%

Disentangling primer interactions improves SARS-CoV-2 genome sequencing by the ARTIC Network’s multiplex PCR

Itokawa

Sekizuka

Hashino

et al. 2020

Preprint

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A group of biologists, ARTIC Network, has proposed a multiplexed PCR primer set for whole genome analysis of the novel corona virus, SARS-CoV-2, soon after the epidemics of this pathogen was revealed. The primer set seems to have been adapted already by many researchers worldwide and contributed for the high-quality and prompt genome epidemiology of this potential pandemic virus. We have also seen the great performance of their primer set and protocol; the primer set was able to amplify all desired PCR products with fairy small amplification bias from clinical samples with relatively high viral load. However, we observed acute drop of reads derived from two particular PCR products, 18 and 76, out of the 98 designated products as sample's viral load decreases. We suspected the reason for this low

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nCoV-2019 sequencing protocol v1

Cited by 340 publications

References 0 publications

Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation During a Pandemic

Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation During a Pandemic

Coast-to-coast spread of SARS-CoV-2 in the United States revealed by genomic epidemiology

Disentangling primer interactions improves SARS-CoV-2 genome sequencing by the ARTIC Network’s multiplex PCR

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