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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Appendix C 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. Sample Informed Consent Form Template (Appendix C of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io dx.doi.org/10.17504/protocols.io.bj6dkra6 Copy
Authors: Giana Schena, Emma Murray
Group: Coronavirus Method Development Community
Summary: During the height of the COVID-19 outbreak, personal protective equipment shortages are felt around the world, in spite of the fact that traditional face masks are not even sufficient to fully protect against the 0.125 micron SARS-CoV-2 virus. Medical personnel, more than any other sector, are being hit hardest by this shortage, given their close contact with infected persons. During this time of crisis and shortage, alternative and atypical solutions are necessary to ensure the protection of those who are on the front lines against this disease. One solution is the creation of reusable 3D-printed personal protective equipment (PPE) in conjunction with appropriate disposable filtration.3D-printed face masks are available on a variety of different open source 3D-printing online platforms. Unfortunately, the general public remains largely unaware of these potential solutions and can also be intimidated by the lack of information available regarding how to integrate scientifically appropriate filters. The intention of this white paper is to make available the information needed for simple construction of effective 3D-printed masks. We recommend a 3D printed face mask design made available by CREALITY, a leader in 3D-printing machines. By using CREALITY’s open source designs and easily obtainable air filters, cut to a desired size, we can increase our ability to protect medical workers against this disease. Additionally, CREALITY offers designs for a 3D-printed eye mask that can be used with plexiglass to protect eyes against airborne droplets. In practice, CREALITY’s 3D model object STL files can be uploaded into any available slicer software allowing for creation of specific commands for 3D printers. Within the slicer, face mask and eye mask models can be scaled to small, medium, or large to fit a variety of face sizes. We have also included strap adjustment clip models for use with facemasks and a face shield to extend the life of traditional facemasks.We also propose a plan to coordinate large-scale construction of crowd-sourced 3D printed masks with collection and delivery of said masks to at-risk businesses, in particular hospitals and care facilities.
Proper citation: Giana Schena, Emma Murray 2020. 3D Printing Of Personal Protective Equipment And Repurposing Of Common Household Air Filters. protocols.io dx.doi.org/10.17504/protocols.io.bd77i9rn Copy
Authors: John Warner, Rebecca Silveston, Delphine Dean
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, Accessible Health
Proper citation: John Warner, Rebecca Silveston, Delphine Dean 2020. Accessible Health Sentinal Test Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bk5wky7e Copy
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 dx.doi.org/10.17504/protocols.io.bdcpi2vn Copy
Authors: Nicola O'Reilly, Svend Kjaer, Maria Greco
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 TET buffer for the RNA extraction protocol.
Proper citation: Nicola O'Reilly, Svend Kjaer, Maria Greco 2020. Preparation of TET Buffer. protocols.io dx.doi.org/10.17504/protocols.io.bfeujjew Copy
Authors: Peter Szabo, Steven Wells, Peter A. Sims, Donna Farber
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: This protocol describes the isolation of lymphocytes and pan-mononuclear cells from human whole blood for scRNAseq analysis.
Proper citation: Peter Szabo, Steven Wells, Peter A. Sims, Donna Farber 2020. COVID Blood Processing for scRNAseq. protocols.io dx.doi.org/10.17504/protocols.io.bjm6kk9e Copy
Authors: Bei Wang, Wen-Hsin Sandy Lee, Cheng-I Wang
Group: Coronavirus Method Development Community
Proper citation: Bei Wang, Wen-Hsin Sandy Lee, Cheng-I Wang 2020. Antibody neutralization assay with SARS-CoV-2 and SARS-CoV pseudovirus. protocols.io dx.doi.org/10.17504/protocols.io.biztkf6n Copy
Authors: Aaron Bivins, Warish Ahmed, Devin North, Kyle Bibby
Group: Coronavirus Method Development Community, 2019-nCoV Wastewater Epidemiology
Summary: The following protocol describes the GERM Lab workflow for processing wastewater for SARS-CoV-2 via pH adjustment, MCE filtration, extraction, and assay by droplet digital PCR (ddPCR). It is synonymous to method A as described in Ahmed et al. 2020 (the referenced manuscript) with modifications to the consumables and PCR format. It is intended for use in a BSL2+ lab with extra precautions for processing environmental specimens for SARS-CoV-2 as described in the CDC Interim Laboratory Biosafety Guidelines.
Proper citation: Aaron Bivins, Warish Ahmed, Devin North, Kyle Bibby 2020. Wastewater Concentration by Adsorption and Direct Extraction for SARS-CoV-2 RNA Detection and Quantification using RT-ddPCR. protocols.io dx.doi.org/10.17504/protocols.io.bhiuj4ew Copy
Authors: Megan Folkerts, mnguyen , Danielle Vazquez, Amber Jones
Group: Coronavirus Method Development Community
Proper citation: Megan Folkerts, mnguyen , Danielle Vazquez, Amber Jones 2020. High-Throughput Tiled Amplicon Sequencing of Sars-CoV2 using Seqwell's plexWell 384. protocols.io dx.doi.org/10.17504/protocols.io.bnkimcue Copy
Authors: Adélaïde Roguet, Shuchen Feng
Group: Coronavirus Method Development Community, mclellan lab
Proper citation: Adélaïde Roguet, Shuchen Feng 2021. Nucleic acid extraction - RNeasy PowerMicrobiome Kit (Qiagen). protocols.io dx.doi.org/10.17504/protocols.io.bpg5mjy6 Copy
Authors: Hsiao-Chung Tsai
Group: Coronavirus Method Development Community, ProtectLife-AnTaimmu, XPRIZE Rapid Covid Testing
Proper citation: Hsiao-Chung Tsai 2020. ENNOLIFE SARS-CoV-2 Antigen Test Kit Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bke3ktgn Copy
Authors: Salvatore D Morgera, Tiffany Miller, Arash Takshi, Stephen E Saddow, Matthew Palm
Group: Coronavirus Method Development Community, Front Line Technologies
Summary: The magnitude of how rapidly the COVID-19 virus could spread and infect others facilitated the need for a rapid COVID-19 test, accessible and affordable for both children and adults worldwide. As a result, an infected patient can quickly quarantine and isolate to slow the spread. Our research group has determined VOCs associated with COVID-19 and has built a prototype gas analysis system similar to a breathalyzer for detecting gasses of COVID-19. Some of the symptoms like headaches, coughing, and diarrhea result in an inflammatory response to create a unique variety of gasses like acetone, hydrogen, and carbon monoxide that can be detected when they are exhaled through a person’s breath. A sample of a person’s breath is first collected in a chamber when a patient breaths into the valve, the gas sensors then detect a voltage change when they contact a COVID-19 target gas. The concentration in parts-per-million is calculated based on the non-linear resistance ratio of the target gas and clean air. Log-based scale calculations and post-data processing calibrate the sensors to increase the accuracy and selectivity. Finally, the voltages and PPM of the target gas concentration data is stored in a computer database and sensor coding reveals the resulting breath pattern signature of COVID-19. At just under $15.00 United States Dollars (USD), test results are just a breath away, within seconds of taking the test and even before leaving the doctor’s office. Although, this system may also be modified to sniff out other pathogens of disease, spoiled meats, and ripened fruit, we are dedicated to build a gas sensor platform for low cost, noninvasive, and rapid detection of the breath pattern signature of COVID-19, in an attempt to slow the spread of the global health threat of infection and in turn to help save lives.Breath analysis systems serve as a noninvasive means for disease screening. Our objective is to utilize this prototype to obtain a database of H2, CO, NO, Acetone and Alcohol concentrations, whereby, COVID patient breath samples are compared to healthy individual breath samples using our breath analysis system prototype. This system is configured for measuring the aforementioned gas concentrations in ppm and recording the data to a text file in a computer database for subsequent analysis. The data obtained from this study is critical to the development of this system.
Proper citation: Salvatore D Morgera, Tiffany Miller, Arash Takshi, Stephen E Saddow, Matthew Palm 2020. Breath Analysis System for Detecting Breath Pattern Signature of COVID-19 . protocols.io dx.doi.org/10.17504/protocols.io.bpcqmivw Copy
Authors: Mickey Shah, Gary Niehaus
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: The Crystal Diagnostics AutoXpress provides rapid detection of COVID 19 at 10 - 100 CFU per ul of clinical sample (nasal swab, sputum, or saliva). The patented Liquid Crystal technology is combined with antibody-coated paramagnetic microspheres to selectively capture and detect COVID 19.
Proper citation: Mickey Shah, Gary Niehaus 2020. CDx autoXpress detection of COVID 19. protocols.io dx.doi.org/10.17504/protocols.io.bki4kugw Copy
Authors: Sarah Reiling, Anne-Marie Roy, Shu-Huang Chen, Josh Quick, Ioannis Ragoussis
Group: Coronavirus Method Development Community, McGill Genome Centre
Summary: Artic nCoV-2019 McGill modified Lunascript Reverse Transcriptase nanopore sequencing protocol.
Proper citation: Sarah Reiling, Anne-Marie Roy, Shu-Huang Chen, Josh Quick, Ioannis Ragoussis 2020. nCoV-2019 McGill Nanopore LibPrep Protocol, 5 ng NB. protocols.io dx.doi.org/10.17504/protocols.io.bjkvkkw6 Copy
Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 5 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. Study Vaccine (Part 5 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io dx.doi.org/10.17504/protocols.io.bj5zkq76 Copy
Authors: Judy Northill, Ian Mackay
Group: protocols.io Ambassadors, 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 case 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 (Wuhan-ORF1ab; 2019-nCoV-related test). protocols.io dx.doi.org/10.17504/protocols.io.bchvit66 Copy
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.Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx.Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers.
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 dx.doi.org/10.17504/protocols.io.bkjgkujw Copy
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 dx.doi.org/10.17504/protocols.io.bg2tjyen Copy
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 dx.doi.org/10.17504/protocols.io.bd5di826 Copy
Authors: Jernej Turnsek
Group: Coronavirus Method Development Community
Summary: Wearing a face mask helps stop the spread of the new coronavirus. This protocol describes a very simple way to make one at home using a cotton pillowcase, 2 rubber bands, and a minute of your time.
Proper citation: Jernej Turnsek 2020. Pillowcase & rubber band DIY face mask. protocols.io dx.doi.org/10.17504/protocols.io.bemjjc4n Copy
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