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Authors: New England Biolabs
Group: Coronavirus Method Development Community
Summary: This protocol details methods for the NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®), NEB #E7660S/L 24/96 reactions.

Proper citation: New England Biolabs 2021. NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) E7660. protocols.io https://dx.doi.org/10.17504/protocols.io.btcenite Copy   

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Authors: Josh Quick
Group: Diaz-Munoz Lab, Coronavirus Method Development Community, Szentagothai Research Centre Virology
Summary: This is a subprotocol for performing adapter ligation with AMII

Proper citation: Josh Quick 2020. Adapter ligation with AMII. protocols.io https://dx.doi.org/10.17504/protocols.io.bdp9i5r6 Copy   

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Authors: Isabel Gautreau
Group: New England Biolabs (NEB), Coronavirus Method Development Community
Summary: This protocol is about the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®).

Proper citation: Isabel Gautreau 2021. NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) (NEB #E7650S/L). protocols.io https://dx.doi.org/10.17504/protocols.io.bsy4nfyw Copy   

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Authors: Shripal Gandhi, Santo Purnama, Keyur Patel, Praveen Sukumara, Dr Rinu R Ravi
Group: Coronavirus Method Development Community, SensingSelfMission, XPRIZE Rapid Covid Testing
Summary: Summary:The S1 COVID-19 Rapid Antigen test kit developed by Sensing Self Pte. Ltd. can quickly and qualitatively detect the spike glycoprotein of novel coronavirus (SARS-COV-2) in human saliva/sputum/stool samples. It can be used as an aid for COVID-19 diagnosis.Objective:According to the clinical trial plan, the S1 COVID-19 Rapid Antigen test kit or “test reagent”, is to test saliva/sputum/stool samples from healthy subjects and confirmed COVID-19 patients. Test results are compared with another commercial SARS-COV-2 nucleic acid detection kit with CFDA approval, which is defined as the “gold standard”. The sensitivity, specificity, and total agreement rate are used to evaluate the feasibility of the test reagent in clinical applications.Method:A collection of clinical samples were examined by the S1 COVID-19 Rapid Antigen test kit and the gold standard SARS-COV-2 nucleic acid detection kit in parallel, to calculate the clinical sensitivity, clinical specificity and total agreement rate of the test reagent.Results:Compared to the gold standard, the clinical sensitivity of test reagent reached 90.0%, the clinical specificity reached 100.0%, and the total coincidence rate reached 95.0%.Conclusions:The performance of test reagent has a high agreement rate with the gold standard, proving its good feasibility in diagnosing suspected COVID-19 cases.Some of the sample collection steps were adapted from the published protocol:https://www.protocols.io/view/saliva-collection-and-rna-extraction-for-sars-cov-bh6mj9c6

Proper citation: Shripal Gandhi, Santo Purnama, Keyur Patel, Praveen Sukumara, Dr Rinu R Ravi 2020. SensingSelf S1 Rapid Antigen Test (Saliva/Sputum/Stool). protocols.io https://dx.doi.org/10.17504/protocols.io.bpudmns6 Copy   

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Authors: Vbarua , Md Ariful Islam Juel, Neha Mittal, Nick Stark, Cynthia Gibas, jschluet , Mariya Munir
Group: Coronavirus Method Development Community, UNC Charlotte COVID WBE

Proper citation: Vbarua , Md Ariful Islam Juel, Neha Mittal, Nick Stark, Cynthia Gibas, jschluet , Mariya Munir 2021. HA Filtration of Wastewater for SARS-CoV-2 detection. protocols.io https:// Copy   

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Authors: peijun he
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, Highfield Diagnostics
Summary: Coronavirus Lateral Flow Assay (LFA) operation protocol

Proper citation: peijun he 2020. Coronavirus Lateral Flow Assay (LFA) operation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bksjkwcn Copy   

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Authors: Julia Borden
Group: Coronavirus Method Development Community
Summary: This protocol describes how to make a face mask out of a t-shirt in 10 easy steps, no sewing required!

Proper citation: Julia Borden 2020. No Sew Face Mask in 10 Steps. protocols.io https://dx.doi.org/10.17504/protocols.io.be2jjgcn Copy   

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Authors: Mahe Raccaud, Manon Garzuel, Joerg Ziegler, Luc Gervais
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: Our capillary-driven microfluidic chips automatically detect multiple SARS-CoV-2 antigens after the addition of a few drops of saliva, triggering a cascade of events powered by capillary forces. We integrate biochemical reagents directly on the microfluidic chips, avoiding the need for peripheral equipment for fluid control. Thus, detection of SARS-CoV-2 can be done at the patient’s side by simply adding sample to the chips, with minimal hands-on time, faster results, and increased sensitivity of fluorescence assays. The use of saliva as a sample matrix avoids painful sample-collection procedures and the need for trained medical staff.

Proper citation: Mahe Raccaud, Manon Garzuel, Joerg Ziegler, Luc Gervais 2020. 1DROP SARS-CoV-2 antigen test. protocols.io https://dx.doi.org/10.17504/protocols.io.bk38kyrw Copy   

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Authors: Ben Lepene, Anurag Patnaik, Robert Barclay
Group: Coronavirus Method Development Community, Nanotrap Applications

Proper citation: Ben Lepene, Anurag Patnaik, Robert Barclay 2020. Capture and concentration of SARS-CoV-2 and other respiratory viruses from VTM / UTM samples using Magnetic Nanotrap® particles for direct RNA extraction. protocols.io https:// Copy   

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Authors: Efthymios Fidanis, Maria Greco, Amelia Edwards, Margaret Crawford, Laura Cubitt, Sophia Ward, Robert Goldstone, Nnenna Kanu, James MacRae, Michael Hubank, Jerome Nicod
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 extraction of RNA from the inactivated sample.Principles of ExaminationThis procedure involves extraction of COVID-19 RNA from inactivated virus samples that have been aliquoted into plates. This protocol follows a method developed in-house that is automated on the Biomek FX liquid handling platform. All reagents and buffers prepared in house are batch tested and certified.

Proper citation: Efthymios Fidanis, Maria Greco, Amelia Edwards, Margaret Crawford, Laura Cubitt, Sophia Ward, Robert Goldstone, Nnenna Kanu, James MacRae, Michael Hubank, Jerome Nicod 2020. Kit-free automated RNA extraction for SARS-CoV-2 testing. protocols.io https://dx.doi.org/10.17504/protocols.io.bfb5jiq6 Copy   

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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 12 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of Vaccine Candidate against COVID-19 in adults > 18 years of age"This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.Please note that this is an evolving document, to be versioned and updated, based on community feedback and new data.

Proper citation: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores 2020. Financing and Insurance (Part 12 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bj59kq96 Copy   

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Authors: Ruth Timme, Heather Blankenship, Erin Young, Emma Griffiths, Duncan MacCannell, Stacia Wyman
Group: GenomeTrakr, Coronavirus Method Development Community, PHA4GE
Summary: PURPOSE: This protocol covers the steps for submitting a SARS-CoV-2 assembly to NCBI's GenBankFor new submitters, there's quite a bit of groundwork that needs to be established before a laboratory can start its first data submission. We recommend that one person in the laboratory take a few days to get everything set up in advance of when you expect to do your first data submission. Two protocols cover the PHA4GE guidance for SARS-CoV-2 submission to NCBI (Raw sequence data, metadata, and assemblies). If you need a pipeline for frequent or large volume submissions, follow Step 1 in the SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject to get your NCBI submission environment established, then contact [email protected] to set up an account for submitting through the API. These protocols cover submission using NCBI's Submission Portal web-interface.Complete in order (1 then 2):1. SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProjectStep-by-step instructions for establishing a new NCBI laboratory submission account and for creating and linking a new BioProject to an existing umbrella effort.Submit SARS-CoV-2 raw data to SRA (Sequence Read Archive) and metadata to BioSample.2. SARS-CoV-2 NCBI assembly submission protocol: GenBank (included protocol) Required: established BioProject and BioSamplesSubmit SARS-CoV-2 consensus sequences to NCBI GenBank, linking to existing BioProject, BioSamples, and raw data.2. SARS-CoV-2 NCBI assembly submission protocol: GenBank (included protocol) Required: established BioProject and BioSamples

Proper citation: Ruth Timme, Heather Blankenship, Erin Young, Emma Griffiths, Duncan MacCannell, Stacia Wyman 2020. SARS-CoV-2 NCBI assembly submission protocol: GenBank. protocols.io https://dx.doi.org/10.17504/protocols.io.bg2tjyen Copy   

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Authors: Mammoth Biosciences, James P. Broughton, Wayne Deng, Clare L. Fasching, Jasmeet Singh, Charles Y. Chiu, Janice S. Chen
Group: Coronavirus Method Development Community
Summary: ***DISCLAIMER: This protocol has not been approved by the FDA and should not be used as a clinical diagnostic***IntroductionGiven the global health emergency, rapid transmission, and severe respiratory disease associated with the outbreak of the 2019 novel coronavirus (SARS-CoV-2), Mammoth Biosciences has reconfigured our DETECTR platform to rapidly and accurately detect SARS-CoV-2 using a visual lateral flow strip format within 30 minutes from sample to result. To ensure specificity of detection, we selected a high-fidelity CRISPR detection enzyme and designed sets of gRNAs that can either 1) differentiate SARS-CoV-2 or 2) provide multi-coronavirus strain detection. SARS-CoV-2 DETECTR couples CRISPR detection with isothermal pre-amplification using primers based on protocols validated by the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO). Currently in the United States, the CDC SARS-CoV-2 real-time RT-PCR diagnostic panel has a laboratory turnaround time of approximately 4-6 hours, with results that can be delayed for >24 hours after sample collection due to shipping requirements. In addition, these tests are only available in CDC-designated public health laboratories certified to perform high-complexity testing. Mammoth is working to enable point of care testing (POCT) solutions that can be deployed in areas at greatest risk of transmitting SARS-CoV-2 infection, including airports, emergency departments, and local community hospitals, particularly in low-resource countries. Leveraging an “off-the-shelf” strategy to enable practical solutions within a short time frame, we describe here a protocol that is fast ( AB1Specifications2Targets● N-gene (SARS-CoV-2 specific) ● E-gene (SARS-CoV, bat-SARS-like-CoV, and SARS-CoV-2 coronaviruses) ● RNase P (human sample control)3Limit of detection70-300 copies/μl inputAcknowledgements: We thank Vikram Joshi, Nefeli Tsaloglou and Xin Miao for advice and helpful discussions in the preparation of this whitepaper.Conflicts of Interest: JPB, CLF, JS and JSC are employees of Mammoth Biosciences, CYC is on the Scientific Advisory Board of Mammoth Biosciences, and JSC is a co-founder of Mammoth Biosciences. JPB, CLF, JS, CYC and JSC are co-inventors on CRISPR-related technologies.

Proper citation: Mammoth Biosciences, James P. Broughton, Wayne Deng, Clare L. Fasching, Jasmeet Singh, Charles Y. Chiu, Janice S. Chen 2020. A protocol for rapid detection of the 2019 novel coronavirus SARS-CoV-2 using CRISPR diagnostics: SARS-CoV-2 DETECTR. protocols.io https://dx.doi.org/10.17504/protocols.io.bcmtiu6n Copy   

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Authors: John Daniels
Group: Coronavirus Method Development Community, Front Line Technologies, XPRIZE Rapid Covid Testing, Team Exhaled Breath Condensate

Proper citation: John Daniels 2020. Mask-Based Covid-19 testing system using Exhaled Breath Condensate. protocols.io https:// Copy   

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Authors: Mariateresa De Cesare
Group: Coronavirus Method Development Community, COG-UK
Summary: Viral RNA library prep using SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian followed by a probe-based bait capture (SeqCap, Roche and xGen, IDT) to generate tagged enriched viral libraries from total RNA that retain directionality in the library. The SMARTer Stranded kit uses random primers, tailed with Illumina Read1 sequence, to start reverse transcription, and a Template Switching Oligo (TSO) to add Read2 sequence at the 3’ of the synthesized cDNA. The 1st stranded cDNA is then amplified using Indexed primers to generate in one step 2nd strand cDNA and complete tagged Illumina libraries. Pooled libraries undergo target enrichment using custom virus-specific biotinylated probes to capture cDNA derived from viral RNA present in the sample.This method has been optimized for large scale viral sequencing projects, and up to 2 x 96-well RNA plates can be prepped in parallel.The protocol follows “Option 2 (without fragmentation)” workflow of the kit User Manual for library preparation, with ¼ of the recommended reaction volume for denaturation and cDNA synthesis, and ½ for PCR, all steps done in 384-well plates.NOTE: the kit contains a Ribodepletion PCR-based module not used in this protocol, so PCR reagents are in excess and a higher volume for PCR can be used safely without depleting the kit of reagents unevenly.The absence of an RNA fragmentation step coupled with more stringent cleanups (at 0.68x) generates libraries with longer insert size, therefor this protocol is only to be used on RNA samples of high quality, and unnecessary freeze-thaw of the RNA MUST be avoided. As the RNA extracted from plasma/swabs is below the detection level for QC, if in doubt about the quality of the RNA provided, proceed with library preparation and pooling, but check the size of the libraries before cleanup of the pool as the 0.68X beads ratio recommended in this protocol would remove most of the library if the starting RNA is of lower quality.To maximize RNA input and improve sensitivity of the assay, an RNA concentration step at the start of the procedure has been added, though not necessary to generate libraries.NOTE: the kit contains a Ribodepletion PCR-based module not used in this protocol, so PCR reagents are in excess and a higher volume for PCR can be used safely without depleting the kit of reagents unevenly.The absence of an RNA fragmentation step coupled with more stringent cleanups (at 0.68x) generates libraries with longer insert size, therefor this protocol is only to be used on RNA samples of high quality, and unnecessary freeze-thaw of the RNA MUST be avoided. As the RNA extracted from plasma/swabs is below the detection level for QC, if in doubt about the quality of the RNA provided, proceed with library preparation and pooling, but check the size of the libraries before cleanup of the pool as the 0.68X beads ratio recommended in this protocol would remove most of the library if the starting RNA is of lower quality.To maximize RNA input and improve sensitivity of the assay, an RNA concentration step at the start of the procedure has been added, though not necessary to generate libraries.

Proper citation: Mariateresa De Cesare 2020. Probe-based target enrichment of SARS-CoV-2. protocols.io https://dx.doi.org/10.17504/protocols.io.bd5di826 Copy   

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Authors: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community
Summary: The first section of this protocol details recommendations for collecting saliva for SARS-CoV-2 detection, either by self-collection or healthcare worker-assisted collection.The second section details an extraction-free method for preparation of saliva samples for SARS-CoV-2 RNA detection. This involves the addition of proteinase K to reduce saliva viscosity, followed by a heating step to inactivate the proteinase K before RT-qPCR testing.The third section describes a dualplex RT-qPCR assay for high-throughput SARS-CoV-2 diagnostics. The CDC_N1 (N1) primer/probe set targets the SARS-CoV-2 nucleocapsid gene, and is shown to be sensitive and specific. The human RNase P (RP) primer/probe set is included as an internal sample control - detection of RP at PCR cycle threshold (CT) 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.

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.bh6jj9cn Copy   

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Authors: Guy Aidelberg, Rachel Aronoff
Group: Coronavirus Method Development Community, Reclone.org (The Reagent Collaboration Network), XPRIZE Rapid Covid Testing, Guy Aidelberg
Summary: A protocol for freeze-drying (Lyophilization) of CoronaDetective tests, and more generally any QUASR RT-LAMP reaction.The amounts here are for one 96 well standard PCR plate and for reactions This can be scaled for any amount and size.

Proper citation: Guy Aidelberg, Rachel Aronoff 2020. Freeze-drying (Lyophilization) and manufacturing of Corona Detective assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bpv4mn8w Copy   

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Authors: Erin Young, TOAST CDC, OAMD CDC
Group: Coronavirus Method Development Community

Proper citation: Erin Young, TOAST CDC, OAMD CDC 2021. Cecret Workflow Protocol for SARS-CoV-2 Assembly and Lineage Classification . protocols.io https:// Copy   

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Authors: Rushna Quddus, Kaitlynn Snyder, Lauren Altman, Laura Philips, David G. Grier, Andrew D. Hollingsworth, Kent Kirshenbaum
Group: Coronavirus Method Development Community, Grier Group, HolographicDiagnostics, XPRIZE Rapid Covid Testing
Summary: This protocol describes the steps required to perform a holographic immunoassay for SARS-CoV-2 whole virus particles. The assay uses an xSight holographic particle characterization instrument (Spheryx, Inc.) to monitor the diameter of specifically functionalized probe beads. The diameter of the beads increases by a few nanometers as targets bind to the surface. xSight detects and reports this change, yielding an estimate for the concentration of analytes in the sample. The method for holographic immunoassays (applied to antibody binding assays) is described inY. Zagzag, M. F. Soddu, A. D. Hollingsworth and D. G. Grier, Holographic molecular binding assays, Scientific Reports 10, 1932 (2020) and K. Snyder, R. Quddus, A. D. Hollingsworth and K. Kirshenbaum, Holographic Immunoassays: Direct Detection of Antibodies Binding to Colloidal Spheres, submitted for publication (2020).

Proper citation: Rushna Quddus, Kaitlynn Snyder, Lauren Altman, Laura Philips, David G. Grier, Andrew D. Hollingsworth, Kent Kirshenbaum 2020. HolographicDiagnostics: Automated Virus Binding Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bkpgkvjw 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) in multiplex PCR. Illumina-compatible sequencing libraries are then made directly from these amplicons in a second PCR step, obviating the need for conventional library preparation. The products of these PCRs are then equivolume pooled and quantitated, prior to sequencing on the Illumina NovaSeq.It is an adaptation of the COVID-19 ARTIC v3 amplicon protocol which can be found here:https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bibtkannBoth the above protocols were adapted from the nCov-2019 sequencing protocol: 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 - tailed method. protocols.io https://dx.doi.org/10.17504/protocols.io.bky5kxy6 Copy   

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