Protocols   Select Another Resource Report Type

Authors: Guerrino Macori, Seamus Fanning
Group: Coronavirus Method Development Community

Proper citation: Guerrino Macori, Seamus Fanning 2020. SARS-CoV-2 Whole Genome Sequencing on Illumina - Tiling PCR. protocols.io https:// Copy   

•   Source:

Authors: Chantal Vogels, Joseph Fauver, Nathan Grubaugh
Group: Coronavirus Method Development Community
Summary: As the B.1.1.7 "UK" variant continues to spread, there is a need for labs to screen COVID-19 samples for these viruses. While sequencing is the gold standard, it cannot always be scaled or implemented in some settings. The Applied Biosystems TaqPath COVID-19 assay (ThermoFisher), a PCR test, was discovered to have a distinct signature (spike gene target failure, [SGTF]) when testing viruses containing the Δ69/70 HV deletion, like the B.1.1.7 variant. Detecting the Δ69/70 HV deletion alone is not definitive for the B.1.1.7 variant, but tracking the frequencies of SGTFs helped the UK track the B.1.1.7 variant. In the US and other countries, screening samples for the SGTF helped to identify potential B.1.1.7 variants for sequencing prioritization. ThermoFisher, however, has not released their spike probe sequence, so the assay needs to be recreated to be used more broadly. Here, we designed a multiplexed PCR assay that recreates the SGTF signature using primer/probe set that targets the Δ69/70 HV deletion, the CDC N1 set as a positive control, and the CDC RNase P set as an extraction/sample control. Samples positive for the CDC N1 set but negative for the Δ69/70 HV deletion set can be tracked and prioritized for sequencing.

Proper citation: Chantal Vogels, Joseph Fauver, Nathan Grubaugh 2021. Multiplexed RT-qPCR to screen for SARS-COV-2 B.1.1.7 variants. protocols.io https://dx.doi.org/10.17504/protocols.io.brcrm2v6 Copy   

•   Source:

Authors: Koen Vandelannoote
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, Koen Vandelannoote
Summary: Here, we describe a semi-automated workflow for paramagnetic bead-based extraction of SARS-CoV-2 RNA from human upper respiratory samples that uses low-cost repurposed Creality3D Ender-3 3D-printers and readily accessible reagents. The protocol described here is for the use of four instruments in parallel, each processing 24 samples, 96 samples per batch.

Proper citation: Koen Vandelannoote 2020. High-throughput SARS-CoV-2 RNA extraction from nasopharyngeal swabs using repurposed 3D printers.. protocols.io https://dx.doi.org/10.17504/protocols.io.bkynkxve Copy   

•   Source:

Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: A real-time RT-PCR to specifically detect SARS-CoV-2 betacoronavirus also called nCoV-2019 or Wuhan seafood market pneumonia virus. Based on sequence MN908947 made available by Professor Yong-Zhen Zhang, Fudan University, Shanghai, China. The target region is within the ORF1ab sequence.NotesAssay is fully optimised (as of 24Jan2020).This test has identified a clinical positive cases of coronavirus disease (COVID-19)

Proper citation: Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR ORF1ab 2020. protocols.io https://dx.doi.org/10.17504/protocols.io.bgtmjwk6 Copy   

•   Source:

Authors: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton
Group: Coronavirus Method Development Community
Summary: The common methods of sample collection for the detection of SARS-Cov2 are either pharyngeal or nasopharyngeal. Both these methods require specific swabs that are in short supply, and both methods are not easily self-administered. A nasal swab can also be used to collect samples and is relatively easy to self-administer, yet obtaining nasal specific swabs can now also be a challenge. In order to address these issues, our group has optimized a protocol for self-administered nasal collection using buccal swabs that are not currently in high demand. With this method, we have been able to collect human samples with reliable results in RNA extraction based on RNaseP, and can detect SARS-Cov2 in known positive cases of COVID-19.

Proper citation: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton 2020. Modified Nasal Swab For Detection of Sars-Cov2. protocols.io https://dx.doi.org/10.17504/protocols.io.bfavjie6 Copy   

•   Source:

Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community
Summary: This is a collection of protocols for: "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. Collection of Protocols and Guidelines for Phase 3 study of Vaccine Candidate for COVID-19. protocols.io https://dx.doi.org/10.17504/protocols.io.bj5pkq5n Copy   

•   Source:

Authors: Moshe Sade-Feldman, Miguel Reyes, Alexandra-Chloe Villani
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: This protocol outlines the steps and methods for blood processing for PBMC to assist the MGH COVID-19 effort.

Proper citation: Moshe Sade-Feldman, Miguel Reyes, Alexandra-Chloe Villani 2020. MGH COVID-19 Effort Blood Processing Protocol for PBMC, Neutrophils and Plasma Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.bjhnkj5e Copy   

•   Source:

Authors: Chantal Vogels, Joseph Fauver, Nathan Grubaugh
Group: Coronavirus Method Development Community
Summary: With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for targeted molecular surveillance methods. While sequencing is the gold standard, it cannot always be immediately scaled or implemented in some settings to detect variants when their frequencies are low. The Applied Biosystems TaqPath COVID-19 assay (ThermoFisher), a PCR test, was discovered to have a distinct signature (spike gene target failure, [SGTF]) when testing viruses containing the Δ69/70 HV deletion, like the B.1.1.7 variant first detected in the UK. However, a sample with a SGTF is not definitive for B.1.1.7, and cannot detect other variants of concern that lack the Δ69/70 HV deletion, such as B.1.351 detected in South Africa and P.1 recently detected in Brazil. We developed a multiplexed RT-qPCR assay that can detect all three variants by targeting the Δ3675-3677 SGF deletion in the ORF1a gene, which has not yet been widely detected in other SARS-CoV-2 lineages. Furthermore, by also targeting the Δ69/70 HV deletion in the spike gene, our assay can differentiate B.1.1.7 from B.1.351 and P.1. Finally, we include the CDC N1 primer and probe set in our multiplexed assay as a control to ensure that target failures are likely due to the presence of the ORF1a and/or spike deletions and that there is sufficient virus RNA for sequencing confirmation. Our multiplexed RT-qPCR assay can be rapidly scaled to support SARS-CoV-2 variant surveillance. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Chantal Vogels, Joseph Fauver, Nathan Grubaugh 2021. Multiplexed RT-qPCR to screen for SARS-COV-2 B.1.1.7, B.1.351, and P.1 variants of concern. protocols.io https://dx.doi.org/10.17504/protocols.io.brrhm536 Copy   

•   Source:

Authors: dkt
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing

Proper citation: dkt 2020. u-Smell-it olfactory test  . protocols.io https://dx.doi.org/10.17504/protocols.io.bkxkkxkw Copy   

•   Source:

Authors: Don Cooper
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing

Proper citation: Don Cooper 2020. COVIDscanDX LAMP. protocols.io https://dx.doi.org/10.17504/protocols.io.bk4dkys6 Copy   

•   Source:

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.bgxjjxkn Copy   

•   Source:

Authors: Ruth Timme, Emma Griffiths, Heather Blankenship, Erin Young, Duncan MacCannell, Stacia Wyman
Group: GenomeTrakr, Coronavirus Method Development Community, PHA4GE
Summary: PURPOSE: This is a SARS-CoV-2 specific protocol that covers the steps needed to establish a new NCBI submission environment for your laboratory, including the creation of new BioProject(s) and submission groups. Once these are step up, the protocol then walks through the process for submitting raw reads to SRA and sample metadata to BioSample through the Submission portal. For 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. 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 BioProject (included protocol)Step-by-step instructions for establishing a new NCBI laboratory submission account and for creating and linking a new BioProject to an existing umbrella effort.SARS-CoV-2 raw data submission to SRA (Sequence Read Archive) and metadata to BioSample.2. SARS-CoV-2 NCBI assembly submission protocol: GenBank Required: established BioProject and BioSamplesSubmit SARS-CoV-2 assemblies to NCBI GenBank, linking to existing BioProject, BioSamples, and raw data.2. SARS-CoV-2 NCBI assembly submission protocol: GenBank Required: established BioProject and BioSamples

Proper citation: Ruth Timme, Emma Griffiths, Heather Blankenship, Erin Young, Duncan MacCannell, Stacia Wyman 2020. SARS-CoV-2 NCBI submission protocol: SRA, BioSample, and BioProject. protocols.io https://dx.doi.org/10.17504/protocols.io.bf7bjrin Copy   

•   Source:

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 S4 Multiplex Covid-19 /MERS-CoV/ Influenza A/B Rapid Antigens Test Kit developed by Sensing Self Pte. Ltd. can quickly and qualitatively detect Novel Coronavirus / Influenza A/ Influenza B /MERS in human saliva/sputum/stool samples. It can be used as an aid for COVID-19 diagnosis. It provides an aid in the diagnosis of infection by the above viruses.Objective:According to the clinical trial plan, the S4 Multiplex Covid-19 /MERS-CoV/ Influenza A/B Rapid Antigen test kit or “test reagent”, is to test saliva/sputum/stool samples from healthy subjects and confirmed patients with infections caused by these viruses. Test results are compared with another commercial Covid-19 /MERS-CoV/ Influenza A/B nucleic acid detection kit, 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 S4 Multplex Covid-19 /MERS-CoV/ Influenza A/B Rapid Antigen test kit and the gold standard SARS-COV-2/MERS-CoV/ Influenza A/B nucleic acid detection kit in parallel, to calculate the clinical sensitivity, clinical specificity and total agreement rate of the test reagent.Results:For SARS-CoV-2, compared to the gold standard:In Saliva, the clinical sensitivity of test reagent reached 88.3%, the clinical specificity reached 100.0%In Sputum, the clinical sensitivity of test reagent reached 93.3%, the clinical specificity reached 100.0% In Stool, the clinical sensitivity of test reagent reached 93.3%, the clinical specificity reached 100.0%For MERS, compared to the gold standard:In Saliva, the clinical sensitivity of test reagent reached 86%, the clinical specificity reached 100.0%In Sputum, the clinical sensitivity of test reagent reached 90%, the clinical specificity reached 100.0% In Stool, the clinical sensitivity of test reagent reached 90%, the clinical specificity reached 100.0%For Influenza Virus A, compared to the gold standard:In Saliva, the clinical sensitivity of test reagent reached 85.7%, the clinical specificity reached 98.3%In Sputum, the clinical sensitivity of test reagent reached 91.4%, the clinical specificity reached 100.0% In Stool, the clinical sensitivity of test reagent reached 90%, the clinical specificity reached 100.0%For Influenza Virus B, compared to the gold standard:In Saliva, the clinical sensitivity of test reagent reached 82.9%, the clinical specificity reached 100.0%In Sputum, the clinical sensitivity of test reagent reached 87.1%, the clinical specificity reached 96.7% In Stool, the clinical sensitivity of test reagent reached 85.7%, the clinical specificity reached 98.3%Conclusions:The performance of test reagent has a high agreement rate with the gold standard, proving its good feasibility in diagnosing suspected Covid-19 /MERS-CoV/ Influenza A/B cases.

Proper citation: Shripal Gandhi, Santo Purnama, Keyur Patel, Praveen Sukumara, Dr Rinu R Ravi 2020. SensingSelf S4 Multiplex Covid-19 /MERS-CoV/ Influenza A/B Rapid Antigens Test Kit (Saliva/Sputum/Stool). protocols.io https://dx.doi.org/10.17504/protocols.io.bkwckxaw Copy   

•   Source:

Authors: Brett Rasile, Kendra Maas
Group: Coronavirus Method Development Community, 2019-nCoV Wastewater Epidemiology, UConn Mars
Summary: Process

Proper citation: Brett Rasile, Kendra Maas 2021. SARS-CoV-2 Wastewater RNA Concentration and Extraction (Nanotrap® and NucleoMag® RNA Water). protocols.io https://dx.doi.org/10.17504/protocols.io.bn58mg9w Copy   

•   Source:

Authors: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton
Group: Coronavirus Method Development Community
Summary: Viral RNA isolation kits used in PCR based tests for the novel coronavirus (SARS-Cov2) are in short supply. Our group sought to identify a method to isolate viral RNA without the use of a kit or other supplies in high demand by clinical labs. Using a TRIzol based RNA extraction, followed by a glycogen precipitation, we are able to isolate a sufficient quality and quantity of RNA for quantitative PCR, and/or droplet digital PCR. This method has been confirmed to work as well as a viral RNA isolation kit, shown to work with SARS-Cov2 RNA-spiked into human samples, and in detecting SARS-Cov2 in known positive cases of COVID-19.

Proper citation: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton 2020. Isolation of SARS-Cov2 RNA from Humans Without High Demand Reagents. protocols.io https://dx.doi.org/10.17504/protocols.io.be8ujhww Copy   

•   Source:

Authors: Gianpaolo Coro
Group: Coronavirus Method Development Community
Summary: This image reports a Maximum Entropy model that estimates suitable locations for COVID-19 spread, i.e. places that could favour the spread of the virus just in terms of environmental parameters.The model was trained just on locations in Italy that have reported a rate of new infections higher than the geometric mean of all Italian infection rates. The following environmental parameters were used, which are correlated to those used by other studies:Average Annual Surface Air Temperature in 2018 (NASA)Average Annual Precipitation in 2018 (NASA)CO2 emission (natural+artificial) averaged between January 1979 and December 2013 (Copernicus Atmosphere Monitoring Service)Elevation (NOAA ETOPO2)Population per 0.5° cell (NASA Gridded Population of the World)The model file (in ASC format) and all parameters used are attached.A higher resolution map and also the model file (in ASC format) and all parameters are available at the external link (Zenodo).The model indicates highest correlation with infection rate for CO2 around 0.03 gCm^−2day^−1, for Temperature around 11.8 °C, and for Precipitation around 0.3 kg m^-2  s^-1, whereas Elevation and Population density are poorly correlated with infection rate.One interesting result is that the model indicates, among others, the Hubei region in China as a high-probability location, and Iran (around Teheran) as a suited location for virus' spread, but the model was not trained on these regions, i.e. it did not know about the actual spread in these regions.Evaluation:A risk score was calculated for each country/region reported by the JHO monitoring system (https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html). This score is calculated as the summed normalised probability in the populated locations divided by their total surface. This score represents how much the zone would potentially foster the virus' spread.We assessed the reliability of this score, by selecting the country/regions that reported thehighest rates of infection. These zones were selected as those with a rate higher than the upper confidence of a log-normal distribution of the rates.The agreement between the two maps (covid_high_rate_vs_high_risk.png, where violet dots indicatehigh infection ratesand countries' colours indicate estimatedhigh risk score) is the following:Accuracy(overall percentage of correctly predicted high-rate zones): 77.25%Kappa(agreement between the two maps):0.46(Good, according to Fleiss' intepretation of the score) This assessment demonstrates that our map can be used to estimate the risk of a certain country to have a high rate of infection, and indicates that the influence of environmental parameters on virus's spread should be further investigated.FilesName (Size) (5.7 MB) md5:dea4e66a1c66d0dfc3b0872adfaa020f (47.3 MB) md5:069727a6c5656d276c475606c9b96d47 (1.9 MB) md5:ca91c4d56654b77bf572eef1a42af7a5 (5.1 MB) md5:0ed217e20ab32aad4ab96e5403670ee4 (2.8 MB) md5:79639fd3540c68450d86fde288edb264 (4.6 MB)md5:57aa6c172b3fc036c08d0560f01436ba (5.5 MB) md5:3ab587ea0e0fbe3fcbd9ea6b7844271a (4.7 MB) md5:7ea930f59e5ff627a18383f02737f78dReferences

Proper citation: Gianpaolo Coro 2020. Suitability Map of COVID-19 Virus Spread. protocols.io https://dx.doi.org/10.17504/protocols.io.bd88i9zw Copy   

•   Source:

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

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

•   Source:

Authors: New England Biolabs
Group: New England Biolabs (NEB), 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®) E7760. protocols.io https://dx.doi.org/10.17504/protocols.io.bsbrnam6 Copy   

•   Source:

Authors: Nikki Freed, Olin Silander
Group: Coronavirus Method Development Community
Summary: To enable faster, easier sequencing of SARS-COV2 genomes with fewer steps than current methods, we use multiplexed 1200 base pair PCR amplicons with the Oxford Nanopore RAPID barcoding kit (RBK004). This is a modification of the ARTIC amplicon V3 sequencing protocol for MinION for nCoV-2019 developed by Josh Quick, which produces 400 base pair amplicons and uses the Oxford Nanopore Ligation barcoding kit (LSK-109). We have increased the size of the amplicons to 1200bp and use the RAPID barcode kit (RBK004), which enables requires less time and fewer reagents than the LSK-109 protocol. The amplicons produced in this protocol could also be used for Illumina sequencing. Primers were all designed using Primal Scheme: http://primal.zibraproject.org/, described here https://www.nature.com/articles/nprot.2017.066.Primer sequences are here: https://docs.google.com/spreadsheets/d/1M5I_C56ZC8_2Ycgm9EFieVlVNqxsP7dXAnGoBZy3nDo/edit?usp=sharingThe primer scheme .bed and .tsv files necessary for the ARTIC variant calling pipeline are at Zenodo: https://zenodo.org/record/3897530#.Xv5EFpMzadYVersion history: V4: updated .bed and .tsv file link to point to Zenodo (and not google drive). V1-V3: included primers sequences in the protocol, fixed step 17.12 from elute in "molecular grade water or Elution buffer" to elute in "10 mM Tris-HCl pH 8.0 with 50 mM NaCl", as suggested on the Oxford Nanopore protocol, changed images from ARTIC protocol image to our own.

Proper citation: Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon). protocols.io https://dx.doi.org/10.17504/protocols.io.bh7hj9j6 Copy   

•   Source:

Authors: Phase Diagnostics
Group: Coronavirus Method Development Community, Phase Diagnostics

Proper citation: Phase Diagnostics 2020. INDICAID Rapid Point-of-Care Test for SARS-CoV-2 Antigen. protocols.io https://dx.doi.org/10.17504/protocols.io.bkt4kwqw Copy   

•   Source: