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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 8 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. Data Handling and Recordkeeping (Part 8 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bj53kq8n Copy   

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Authors: Driss LAHLOU KITANE, Salma LOUKMAN, Salma LOUKMAN
Group: Coronavirus Method Development Community, Team Spectra

Proper citation: Driss LAHLOU KITANE, Salma LOUKMAN, Salma LOUKMAN 2020. COVID 19 testing using ATR spectrometer and AI.. protocols.io https://dx.doi.org/10.17504/protocols.io.bqfymtpw Copy   

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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 13 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. Publication and Data Sharing Policy (Part 13 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bj6akrae Copy   

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Authors: Diego Lai
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: LooK SPOT COVID-19 antigen rapid test uses a smartphone-based LooK SPOT reader, antigen cassette, and LocationNow AI Cloud to qualitatively detect the SARS-CoV-2 Nucleocapsid protein antigen within 5 minutes. The reagent is used to detect the SARS-CoV-2 nucleocapsid protein antigen, which is usually present in the upper respiratory tract sample in the acute phase of infection. When a positive result is presented, it means that there are viral antigens in the sample. The LooK SPOT system consists of 5 key components: LooK SPOT reader LooK SPOT app LooK COVID-19 antigen cassetteLooK PASS appLocationNow AI CloudTo start the test, the patient has to download and register the LooK PASS app from the app stores. The patient scans the QR code of the LooK COVID-19 antigen cassette with the LooK PASS app, and the information is uploaded to the LocationNow AI Cloud for the registration of the test. Then place the nasopharyngeal swab with the sample of the patient into the Extraction Buffer Tube. Stir the nasopharyngeal swab in the Extraction Buffer Tube and wait for one (1) minute. The virus particles in the sample are disrupted and exposing the internal viral nucleoproteins. Use the liquid dropper to extract the liquid inside the tube and apply three drops in the sample window of the antigen cassette. Insert the antigen cassette into LooK SPOT reader attached to a smartphone by using the camera of the smartphone to take pictures from the sample. LooK SPOT reader detects the ID of the LooK antigen cassette and sends these images of the sample reading to LocationNow AI cloud for analysis. LocationNow AI cloud analyzes the images of the fluorescent signal of the sample by using proprietary AI algorithms and returns results of Positive, Negative, or Invalid to the LooK SPOT reader within 5 minutes. The same result is also sent to the patient's smartphone's LooK PASS app. This test procedure is secured with full privacy. LocationNow AI algorithms have reduced the false-negative cases caused by human visual check errors. LooK COVID-19 antigen rapid test is designed for use at the Point of Entry for the revival of the economy.

Proper citation: Diego Lai 2020. LooK SPOT Antigen Rapid Test System. protocols.io https://dx.doi.org/10.17504/protocols.io.bks6kwhe Copy   

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Authors: Josh Quick
Group: ARTIC, Coronavirus Method Development Community, COG-UK

Proper citation: Josh Quick 2020. nCoV-2019 sequencing protocol v3 (LoCost). protocols.io https:// Copy   

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Authors: Daphna Strauss, Ayelet Rahat, Israa Sharkia, Alon Chappleboim, Miriam Adam, Daniel Kitsberg, Gavriel Fialkoff, Matan Lotem, Omer Gershon, Anna-Kristina Schmidtner, Esther Oiknine-Djian, Agnes Klochendler, Ronen Sadeh, Yuval Dor, Dana Wolf, Naomi Habib, Nir Friedman
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: The global SARS-CoV-2 pandemic led to a steep increase in the need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays that involve a separate RNA extraction and qPCR reaction for each sample with a fixed cost and reaction time. While automation and improved logistics can increase the capacity of these tests, they cannot exceed this lower bound dictated by one extraction and reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information on viral strains and host immune response.Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, ApharSeq (Amplicon Pooling by Hybridization And RNA-Seq), includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one step is performed before pooling hundreds of barcoded samples for subsequent steps and further analysis. Considering these improvements, our proposed workflow is estimated to reduce costs by 10-50 fold, labor by 5-100 fold, automated liquid handling by 5-10 fold, and reagent requirements by 100-1000 fold compared to existing methods.

Proper citation: Daphna Strauss, Ayelet Rahat, Israa Sharkia, Alon Chappleboim, Miriam Adam, Daniel Kitsberg, Gavriel Fialkoff, Matan Lotem, Omer Gershon, Anna-Kristina Schmidtner, Esther Oiknine-Djian, Agnes Klochendler, Ronen Sadeh, Yuval Dor, Dana Wolf, Naomi Habib, Nir Friedman 2020. SARS-CoV-2 detection with ApharSeq. protocols.io https://dx.doi.org/10.17504/protocols.io.bkdcks2w Copy   

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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Appendix B 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. Severity Grading Table (Appendix B of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bj6ckraw 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. V.2 - 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.bjr9km96 Copy   

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Authors: Daryl Gohl
Group: Coronavirus Method Development Community
Summary: This protocol outlines how to process RNA for SARS-CoV-2 sequencing using tailed primers to generate tiled amplicons using the method described here: https://www.biorxiv.org/content/10.1101/2020.05.11.088724v1.Best results are obtained for samples with N1 and N2 Ct values of https://www.biorxiv.org/content/10.1101/2020.04.02.022186v1.full). For samples with N1 and N2 values between 30 and ~35, coverage and other sequencing metrics may be more variable and increased adapter dimer formation is expected.

Proper citation: Daryl Gohl 2020. SARS-CoV-2 Tailed Amplicon Illumina Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bipikdke Copy   

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Authors: The Crick COVID-19 Consortium
Group: Coronavirus Method Development Community, Crick COVID-19 Consortium
Summary: The Crick COVID-19 Consortium commenced on 19 March 2020 in response to the emerging COVID-19 pandemic in a collaboration between University College London Hospitals, Health Services Laboratory (HSL), the Institute of Cancer Research and the Francis Crick Institute.Over 2 weeks, the consortium has set up a high throughput RT-PCR COVID-19 diagnostics assay in an academic environment. The work is conducted at the Francis Crick Institute as an extension of the accredited laboratories at HSL. The aim has been to minimise dependencies on reagents, in short supply globally, to provide resilience to the pipeline and provide a scale-able platform to screen hundreds to thousands of healthcare workers and patients per day.The interactive Standard Operating Procedures (SOP) provide in this collection a step-by-step approach to set up a diagnostic pipeline in an academic institute in collaboration with partner laboratories and hospital trusts.--The members of the Crick COVID-19 Consortium would like to thank Julia Roßmanith, Friedrich-Alexander Universität Erlangen-Nürnberg, for her assistance with the editing of this collection of protocols.

Proper citation: The Crick COVID-19 Consortium 2020. The Crick COVID-19 RT-PCR Testing Pipeline. protocols.io https://dx.doi.org/10.17504/protocols.io.bfe2jjge Copy   

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Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: A real-time RT-PCR designed to amplify a portion of the ORF1ab gene of sequences from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)The probe and primers were published by the China CDC and are now included in the WHO in-house assay document.This test has been modified for use with our standard reagents and instrumentation.This test has identified clinical positive cases of coronavirus disease 2019 (COVID-19).

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

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Authors: Joel Sevinsky, Arian Nassiri, Heather Blankenship, Erin Young, 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 and the Illumina Nexterra DNA Flex library prep kit. 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.Feedback and comments appreciated.

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

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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=sharing

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.bgggjttw Copy   

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Authors: Nabil-Fareed Alikhan, Emma Griffiths, Ruth Timme, Duncan MacCannell
Group: Coronavirus Method Development Community, PHA4GE
Summary: This protocol provides the steps needed to establish a new GISAID submission environment for your laboratory. Once established, this protocol covers genome submission sample meta to GISAID.

Proper citation: Nabil-Fareed Alikhan, Emma Griffiths, Ruth Timme, Duncan MacCannell 2020. SARS-CoV2 GISAID submission protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bh98j99w Copy   

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Authors: Veronica Antelo, Iraola Lab
Group: Coronavirus Method Development Community

Proper citation: Veronica Antelo, Iraola Lab 2020. Sampling and viral concentration for SARS-CoV-2 detection in wastewater . protocols.io https://dx.doi.org/10.17504/protocols.io.bf5mjq46 Copy   

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Authors: Monica Trujillo, Kristen Cheung, Tianhui Gao, Irene Hoxie, Sherin Kannoly, Kaung Myat San, Davida S. Smyth, John Dennehy
Group: Coronavirus Method Development Community
Summary: The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Furthermore, the RNA obtained using this protocol can be sequenced both using a targeted approach sequencing specific regions or the whole genome. The protocol was adopted by the NYC Department of Environmental Protection in August 2020 to support their efforts in monitoring SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. Owing to the pasteurization step, it is safe for use in a BSL1+facility. This step does not reduce the genetic signal of the virus while making the protocol safe for the personnel involved. This protocol could be used to isolate a variety of other clinically relevant viruses from wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.

Proper citation: Monica Trujillo, Kristen Cheung, Tianhui Gao, Irene Hoxie, Sherin Kannoly, Kaung Myat San, Davida S. Smyth, John Dennehy 2021. Protocol for Safe, Affordable, and Reproducible Isolation of SARS-CoV-2 RNA from Wastewater. protocols.io https://dx.doi.org/10.17504/protocols.io.brr6m59e Copy   

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Authors: bkatchman
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: The DetectX-Rvtest is a test based on end-point reverse transcription polymerase chain reaction (RT-PCR) coupled to DNA microarray hybridization for the detection of multiple genes within SARS-CoV-1 and SARS-CoV-2 viruses.The DNA microarray contains:DetectX-Rv Kit – -DetectX-Rv SARS-CoV-2 Multiplex Assay—contains 5 (five) SARS-CoV-2 primer sets; 4 (four) SARS-CoV-2probes targeting each N1, N2, and N3 genes-DetectX-Rv RNase P Control—internal process control for nucleic acid extraction 2 (two) RNase P primer and probe sets as an internal positive control. DetectX-Rv SARS-CoV-2 Control – RNA control that contains targets specific to the SARS-CoV-2 genomic regions that are targeted by the assay-DetectX-Rv SARS-CoV-2 Multiplex Assay—contains 5 (five) SARS-CoV-2 primer sets; 4 (four) SARS-CoV-2probes targeting each N1, N2, and N3 genes-DetectX-Rv RNase P Control—internal process control for nucleic acid extraction 2 (two) RNase P primer and probe sets as an internal positive control.

Proper citation: bkatchman 2020. DetectXRv Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.bkg4ktyw Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB), Coronavirus Method Development Community
Summary: This protocol utilizes the Monarch Total RNA Miniprep Kit to purify RNA from buccal swabs, nasopharyngeal samples, and saliva.

Proper citation: New England Biolabs 2020. RNA Purification from Buccal Swabs, Nasopharyngeal Samples (swab or aspirate) and Saliva using the Monarch Total RNA Miniprep Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.be93jh8n Copy   

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Authors: Stephanie Loeb, Katy Graham, David Catoe, Marlene Wolfe, Alexandria Boehm, Krista Wigginton
Group: Coronavirus Method Development Community, Wastewater-based epidemiology working group
Summary: Goal: Quantify concentration of N1, N2, BCoV, and PMMoV in RNA extractions using RT-ddPCR. 8 hours per RT-ddPCR plate

Proper citation: Stephanie Loeb, Katy Graham, David Catoe, Marlene Wolfe, Alexandria Boehm, Krista Wigginton 2020. One-Step RT-ddPCR for Detection of SARS-CoV-2, Bovine Coronavirus, and PMMoV RNA in RNA Derived from Wastewater or Primary Settled Solids . protocols.io https://dx.doi.org/10.17504/protocols.io.bi6vkhe6 Copy   

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Authors: Sarah Reiling, Anne-Marie Roy, Shu-Huang Chen, Ioannis Ragoussis
Group: Coronavirus Method Development Community, McGill Genome Centre
Summary: This protocol works for 12 native barcodes, 24 native barcodes, and 96 native barcodes.

Proper citation: Sarah Reiling, Anne-Marie Roy, Shu-Huang Chen, Ioannis Ragoussis 2020. nCoV-2019 McGill Nanopore LibPrep Protocol, 10 ng NB. protocols.io https://dx.doi.org/10.17504/protocols.io.bjktkkwn Copy   

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