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Authors: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community
Summary: SalivaDirect is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets.Version 2 includes: Optimized thermocycler conditionsLocally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclersUse of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates.Version 3 has been updated to remove steps for sample self-collection.

Proper citation: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie 2020. SalivaDirect: RNA extraction-free SARS-CoV-2 diagnostics. protocols.io https://dx.doi.org/10.17504/protocols.io.bii4kcgw Copy   

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Authors: Josh Quick
Group: ARTIC, Coronavirus Method Development Community, COG-UK
Summary: ARTIC amplicon sequencing protocol for MinION for nCoV-2019This one-pot native barcoding protocol was developed in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC.

Proper citation: Josh Quick 2020. nCoV-2019 sequencing protocol v2 (GunIt). protocols.io https://dx.doi.org/10.17504/protocols.io.bdp7i5rn Copy   

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Authors: Elias Dahdouh, Fernando Lazaro, Maria Rodriguez Tejedor, Jesus Mingorance
Group: Coronavirus Method Development Community
Summary: This protocol is for the preparation of libraries for 96 samples of SARS-CoV-2 using the New England Biolabs NEBNext®Fast DNA Library Prep Set for Ion Torrent™ kit, ARTIC primer pools, the Ion Express Barcodes, and sequencing through Ion Torrent. The library preparation is semi-automated with the open source Opentrons robot. The volumes, concentrations, cycling conditions, and Opentrons protocols have been optimized through a series of experiments and are currently being used at our lab on a weekly basis. The samples used for this protocol were extracted after being obtained from nasopharyngeal swabs, placed in viral transport medium, and mixed 1:1 with AVL buffer (Qiagen). It is advisable to perform a screening for the samples before starting the library preparation by RT-qPCR and select the samples that have Ct values below 30. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Elias Dahdouh, Fernando Lazaro, Maria Rodriguez Tejedor, Jesus Mingorance 2021. High Throughput Semi-Automated SARS-CoV-2 Library Preparation Protocol for Ion Torrent Sequencing using Opentrons, New England Biolabs Kit, and ARTIC Primers. protocols.io https://dx.doi.org/10.17504/protocols.io.brjfm4jn Copy   

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Authors: Jessica E. Manning, Jennifer Bohl, Sreyngim Lay, Sophana Chea, Vida Ahyong, Erik Karlsson
Group: Chan Zuckerberg Biohub, Coronavirus Method Development Community
Summary: This protocol was used to enrich for SARS-CoV2 sequencing reads from a confirmed COVID-19 swab sample and attain the full genome using an iSeq100. By using a spiked primer approach with 73 primers spanning the entire SARS-CoV2 genome, we were able to get an average of 15x genome coverage on an iSeq100 with 1.8 million paired end-reads. Here we overview all the steps, from sample extraction, library preparation with a spiked primer enrichment step, and sequencing on an iSeq100. The collaborative effort involved the Manning Lab, NIAID in Phnom Penh, Cambodia, Institut Pasteur Cambodge, Cambodia Ministry of Health, the Chan Zuckerberg Biohub, and the Chan Zuckerberg Initiative.This approach was developed based on the work from Deng et al, Nature Microbiology, January 13, 2020. https://www.nature.com/articles/s41564-019-0637-9

Proper citation: Jessica E. Manning, Jennifer Bohl, Sreyngim Lay, Sophana Chea, Vida Ahyong, Erik Karlsson 2020. SARS-CoV-2 Enrichment Sequencing by Spiked Primer MSSPE method. protocols.io https://dx.doi.org/10.17504/protocols.io.bc36iyre Copy   

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Authors: DNA Pipelines R&D, Benjamin Farr, Diana Rajan, Emma Betteridge, Lesley Shirley, Michael Quail, Naomi Park, Nicholas Redshaw, Iraad Bronner, Louise Aigrain, Scott Goodwin, Scott Thurston, Stefanie Lensing, James Bonfield, Keith James, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, David K. Jackson, Alex Alderton, Ian Johnston
Group: Coronavirus Method Development Community, COG-UK
Summary: This SOP describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing 400nt amplicons tiling the viral genome using V3 nCov-2019 primers (ARTIC). This is followed by library construction, equivolume pooling of samples and quantitation, prior to sequencing on the Illumina NovaSeq. It is adapted from the nCov-2019 sequencing protocol which can be found here: https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w.

Proper citation: DNA Pipelines R&D, Benjamin Farr, Diana Rajan, Emma Betteridge, Lesley Shirley, Michael Quail, Naomi Park, Nicholas Redshaw, Iraad Bronner, Louise Aigrain, Scott Goodwin, Scott Thurston, Stefanie Lensing, James Bonfield, Keith James, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, David K. Jackson, Alex Alderton, Ian Johnston 2020. COVID-19 ARTIC v3 Illumina library construction and sequencing protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bgq3jvyn Copy   

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Authors: Nicola O'Reilly
Group: Coronavirus Method Development Community, Crick COVID-19 Consortium
Summary: Purpose of examination / Clinical relevanceAt the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as COVID19. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to the Crick for testing.Principles of ExaminationThis procedure involves the preparation of 0.2M EDTA pH 8.0 for making L6 virus inactivation buffer.

Proper citation: Nicola O'Reilly 2020. Preparation of 0.2M EDTA, pH 8.0. protocols.io https://dx.doi.org/10.17504/protocols.io.bfexjjfn Copy   

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Authors: WHO
Group: Coronavirus Method Development Community
Summary: Composition of alcohol-based formulations for in-house/local production The choice of components for WHO handrubs takes into account both cost constraints and microbiological efficacy. The procurement of raw ingredients will be influenced by the availability of sub-standard materials on the market and it is important to select local sources with care. The following two alcohol-based handrub formulations are recommended for preparation in-house or in a local production facility, up to a maximum of 50 litres: Formulation 1 To produce final concentrations of ethanol 80% v/v, glycerol 1.45% v/v, hydrogen peroxide (H2O2) 0.125% v/v. Formulation 2 To produce final concentrations of isopropyl alcohol 75% v/v, glycerol 1.45% v/v, hydrogen peroxide (H2O2) 0.125% v/v: Only pharmacopoeial quality reagents should be used (e.g. The International Pharmacopoeia) and not technical grade products

Proper citation: WHO 2020. WHO-recommended handrub formulations. protocols.io https://dx.doi.org/10.17504/protocols.io.bdqei5te Copy   

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Authors: vito
Group: Coronavirus Method Development Community
Summary: Qbiotix has developed novel nucleic acid assays and sample preparation procedures to improve global microbial detection standards and rapidly/inexpensively test high volume of samples. Qbiotix aims to enable countries to test their entire populations, their food, their waters with millions of tests per day on a regular basis.The assays are isothermal and homogeneous. Thermocycling is not required and amplification is non-enzymatic removing the need for complex instrumentation and reliance on variable quality enzymes. Critically we have removed the need for complex nucleic acid preparation, integrated the assay into the collection/transport solution and significantly reduced reliance on a complex supply chain. The assays remove a good proportion of the constraints of PCR which heretofore have inhibited the number of people being testing for SARS-CoV-2 in the US and globally.

Proper citation: vito 2020. Qbiotix SARS-CoV-2 protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bkvckw2w Copy   

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Authors: Rabiatul Adawiyah, Patricia Ng, Bei Wang, Cheng-I Wang
Group: Coronavirus Method Development Community

Proper citation: Rabiatul Adawiyah, Patricia Ng, Bei Wang, Cheng-I Wang 2020. Antibody characterizations by BioLayer Interferometry (BLI). protocols.io https://dx.doi.org/10.17504/protocols.io.bi39kgr6 Copy   

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Authors: Anne Berking
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: The "Rapid Ag test based on Alpaca Nanobodies" is a test developed with single domain antibodies. This type of antibody is capable of effectively recognize the Spike protein on the surface of SARS-CoV2 leading a signal that is 10 times stronger than conventional antibodies. It takes only 16 minutes from sample to result. The procedures for using this test were designed to ensure an easy use and interpretation, making it a quick and simple test.

Proper citation: Anne Berking 2020. Rapid column Ag test. protocols.io https://dx.doi.org/10.17504/protocols.io.bqdjms4n Copy   

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Authors: Joel Sevinsky, Arian Nassiri, Erin Young, Heather Blankenship, Kevin Libuit, Kelly Oakeson, Lauren Turner, StaPH-B Consortium
Group: Coronavirus Method Development Community, StaPH-B, Theiagen Consulting LLC
Summary: This protocol is an adaption of several circulating protocols on SARS-CoV-2 sequencing using the ARTIC protocol. Its purpose is to simplify things for the average state public health laboratory, using equipment and expertise they currently posess, most likely from their funded PulseNet activities.This protocol is derived from other works, including:https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6whttps://www.protocols.io/view/ncov-2019-sequencing-protocol-v2-bdp7i5rn

Proper citation: Joel Sevinsky, Arian Nassiri, Erin Young, Heather Blankenship, Kevin Libuit, Kelly Oakeson, Lauren Turner, StaPH-B Consortium 2020. SARS-CoV-2 Sequencing on Illumina MiSeq Using ARTIC Protocol: Part 1 - Tiling PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bfefjjbn Copy   

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Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: A nested RT-PCR targeting the RdRp region of the sub-genus Sarbecovirus. The primers are modified from the pan-coronavirus RT-PCR published by Hu et al. 2017 to be more specific to SARS-CoV-2.Assay may be used in resource poor settings where real-time cyclers are not available.Sanger sequencing can be used to confirm SARS-CoV-2 where WGS is not available or where WGS fails due to poor quality sample.

Proper citation: Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRp nested RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bcmpiu5n Copy   

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Authors: Max Marrone
Group: Coronavirus Method Development Community, Opentrons COVID-19 Testing
Summary: Opentrons and the Open Medicine Institute are developing an automated high-throughput COVID-19 testing protocol to submit to the FDA for an Emergency Use Authorization as a diagnostic.The standard assay for this type of infectious disease testing is quantitative PCR (qPCR), and in this case reverse transcriptase qPCR since COVID-19 is an RNA virus. After patient samples are collected in public health facilities, doctors offices, and hospitals, they are sent to the lab for processing, which happens in four steps:Sample IntakeRNA ExtractionqPCR SetupRT-qPCR AssayOpentrons OT-2 robots carry out most of this work. However, human operators are needed for some key tasks, like:Moving samples between stationsPreparing certain reagentsRunning the qPCR machineLogging data

Proper citation: Max Marrone 2020. Opentrons COVID-19 testing: Stations A & B, BP Genomics kit, 24 samples. protocols.io https://dx.doi.org/10.17504/protocols.io.be48jgzw Copy   

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Authors: Roser Vento-Tormo
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: This protocol details the steps and methods of PBMC purification

Proper citation: Roser Vento-Tormo 2020. Purification of PBMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.bjinkkde Copy   

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Authors: Oscar Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium
Group: Coronavirus Method Development Community
Summary: This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.

Proper citation: Oscar Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium 2020. Direct wastewater RNA capture and purification via the "Sewage, Salt, Silica and SARS-CoV-2 (4S)" method. protocols.io https://dx.doi.org/10.17504/protocols.io.biwekfbe Copy   

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Authors: Christopher Hughes
Group: Coronavirus Method Development Community

Proper citation: Christopher Hughes 2020. High-throughput SARS-CoV-2 and host genome sequencing - alignment protocol . protocols.io https:// Copy   

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Authors: Isabel Ott, Chantal Vogels, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community
Summary: This protocol details recommendations for collecting and processing saliva for SARS-CoV-2 detection, as used in Wyllie et al. 2020, which details results of testing saliva collected from COVID-19+ inpatients and asymptomatic healthcare workers.COVID-19 inpatients. Saliva samples were self-collected by the patient. Upon waking, patients were asked to avoid food, water and brushing of teeth until the sample was collected. Patients were asked to repeatedly spit into a sterile 90 mL specimen collection cup until roughly a quarter full of liquid (excluding bubbles), before securely closing it. For patients unable to provide saliva (such as those on mechanical ventilation), clinical teams were advised that suction could be used to collect saliva into the cup (or a sputum trap container for improved containment and safety). Collected volumes ranged from 0.5 - 20 mL. All saliva samples were stored at room temperature and transported to the research lab at the Yale School of Public Health within 5 hours of collection, with RNA for SARS-CoV-2 detection extracted within 12 hours of collection. When possible, saliva samples were stored at +4°C, otherwise they were kept at room temperature.Asymptomatic healthcare workers. Asymptomatic healthcare workers were asked to collect ~10 mL of saliva into a sterile specimen collection cup. No specific instructions were given regarding food intake etc prior to collection. Samples were delivered to the research lab at the Yale School of Public Health within 6 hours of collection and stored for up to 6 hours at +4°C until aliquoting for RNA extraction. Samples collected on overnight shifts were stored at +4°C before delivery to the research lab.

Proper citation: Isabel Ott, Chantal Vogels, Nathan Grubaugh, Anne Wyllie 2020. Saliva Collection and RNA Extraction for SARS-CoV-2 Detection. protocols.io https://dx.doi.org/10.17504/protocols.io.bh6mj9c6 Copy   

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Authors: Tammy Krylova, Tim Budd, Jerome Nicod, Rupert Beale, Simon Caidan
Group: Coronavirus Method Development Community, Crick COVID-19 Consortium
Summary: Purpose of examination / Clinical relevanceAt the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as SARS-Cov-2. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to the Crick for testing. The first step of the process is sample receipt at the Crick. This SOP describes the inactivation of the virus.

Proper citation: Tammy Krylova, Tim Budd, Jerome Nicod, Rupert Beale, Simon Caidan 2020. Viral inactivation of clinical samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bfadjia6 Copy   

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Authors: Ayelet Rahat, Miriam Adam, Uri Shabi, Moshe Cohen, Daniel Kitsberg, Malka Nissim-Rafinia, Hagit Turm, Agnes Klochendler, Daphna Joseph-Strauss, Israa Sharkia, Matan Lotem, Gavriel Fialkoff, Ronen Sadeh, Alon Chappleboim, Yuval Dor, Nir Friedman, Dana Wolf, Naomi Habib
Group: Coronavirus Method Development Community
Summary: The current SARS-CoV2 epidemic calls for large scale viral tests. The current testing procedure for the presence of virions involves several steps - pharyngeal/nasal swab, cell/viral lysis, RNA extraction, and an rtPCR assay targeting the viral genome and a human gene as an internal sample control. Improvement of any of these steps in terms of cost, processing time, or reagent availability, could result in a significant increase of testing capacity world-wide.Here, we describe a rapid and efficient home made SPRI-based RNA extraction method from a lysed sample. Our approach can be fully automated, is quick (

Proper citation: Ayelet Rahat, Miriam Adam, Uri Shabi, Moshe Cohen, Daniel Kitsberg, Malka Nissim-Rafinia, Hagit Turm, Agnes Klochendler, Daphna Joseph-Strauss, Israa Sharkia, Matan Lotem, Gavriel Fialkoff, Ronen Sadeh, Alon Chappleboim, Yuval Dor, Nir Friedman, Dana Wolf, Naomi Habib 2020. Sars-CoV2 RNA purification with homemade SPRI beads for RT-qPCR test. protocols.io https://dx.doi.org/10.17504/protocols.io.beswjefe Copy   

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Authors: Sam Diaz-Munoz, Ivy Jose, AJ Campbell
Group: Diaz-Munoz Lab, Coronavirus Method Development Community
Summary: ARTIC amplicon MinION sequencing protocol for nCoV-2019 starting from environmental samplesThis protocol is a modification of the nCoV-2019 sequencing protocol v2 (GunIt) V.2, a one-pot native barcoding protocol was developed by Josh Quick in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC. This version modifies a few steps from the above protocol to obtain a better yield from low concentration samples.This protocol was implemented in the manuscript: "Environmental Surveillance for SARS-CoV-2 at the UC Davis Medical Center" by David Coil et al. Code for data analysis here:https://github.com/sociovirology/sars_cov2_environmental_seqThis protocol was developed by the Díaz-Muñoz Lab: Sam Díaz-Muñoz, Ivy José, and A.J. Campbell.

Proper citation: Sam Diaz-Munoz, Ivy Jose, AJ Campbell 2021. nCoV-2019 environmental sample sequencing protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.brnbm5an Copy   

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