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Authors: Josh Quick
Group: Diaz-Munoz Lab, Coronavirus Method Development Community, Szentagothai Research Centre Virology

Proper citation: Josh Quick 2019. DNA quantification using the Quantus fluorometer . protocols.io https://dx.doi.org/10.17504/protocols.io.7pzhmp6 Copy   

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Authors: Lea Starita
Group: Coronavirus Method Development Community
Summary: Molecular Detection of SARS-CoV-2 for SCAN the Greater Seattle Coronavirus Assessment Network .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; } The SCAN real-time RT-PCR assay for SARS-CoV-2 contains primer/probe sets that target the Orf1b and S genes, designed against the SARS-CoV-2 genome (GenBank: MN908947.3). The primer/probe sets were analyzed in silico for specificity for this specific betacoronavirus by Thermo Fisher. All assays are performed on total nucleic acids extracted from upper respiratory tract swabs, including mid-turbinate and nasopharyngeal (NP) swabs stored in Universal Transport Media. The assay limit of detection of 5.6 (Orf1b) and 12.9 (S) molecules per reaction was determined from serial dilutions of synthetic DNA of the target sequences. These probes reproducibly detected SARS-CoV-2 RNA from cultured virus from USA-WA-1/2020 (BEI resources/ATCC) and from 21 specimens confirmed to be positive by the Washington State Department of health. Specificity was determined by lack of detection of SARS-CoV-2 from specimens that are known to be positive for seasonal coronavirus strains HKU1/NL63 or 229E/OC43 and/or other respiratory viruses. These specimens are further inferred to be negative by the fact that they were obtained in the greater Seattle area prior to the first known reports of SARS-CoV-2 in China. .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Lea Starita 2020. COVID-19 SCAN molecular workflow. protocols.io https://dx.doi.org/10.17504/protocols.io.bebkjakw Copy   

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Authors: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda
Group: Coronavirus Method Development Community
Summary: This protocol is folked from "ARTIC amplicon sequencing protocol for MinION for nCoV-2019" by Josh Quick to adapt it to illumina sequencers. Because the PCR products are fragmented and ligated with adapters, this protocol is not restricted to 250 PE mode of MiSeq. While the library preparation uses QiaSeq FX by Qiagen and is basically straight forward (as par kit instruction but set to 1/4 scale), some tweaks for much of simplicity and speed were added.Change from the previous version (V1): The amount of cDNA input to multiplex PCR is increased (now almost same amount to the ARTIC's original protocol) (Step 10). This change gives better results for samples with extremely low RNA copy. Amount of adapter solution input was corrected (Step 19).

Proper citation: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda 2020. nCoV-2019 sequencing protocol for illumina. protocols.io https://dx.doi.org/10.17504/protocols.io.betejeje Copy   

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Authors: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton
Group: Coronavirus Method Development Community
Summary: Assay sensitivity is an important part of any lab test, this becomes a more critical issue when dealing with matters involving public health. The potential of pre- or asymptomatic carriers, as well as convalescents which have not completely cleared the virus are a public health risk. PCR based tests, specifically quantitative PCR is the gold standard to test for the novel coronavirus (SARS-Cov2), however, it is limited by its detection capabilities, and the results are based off of an amplification curve, rather than a real number or concentration. The limit of detection with quantitative PCR could easily yield a false negative for contagious individuals. In comparison, droplet digital PCR (ddPCR) is estimated to be 500 times more sensitive than quantitative PCR, and results are displayed as an absolute number (copies/uL). In this method our group adapted from our usual studies related to Parkinson's disease, we have been able to confirm that ddPCR is more sensitive than quantitative PCR, detect SARS-Cov2 RNA-spiked into human samples, and detect SARS-Cov2 in known positive cases of COVID-19.

Proper citation: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton 2020. Detection of Sars-Cov2 Using Droplet Digital PCR . protocols.io https://dx.doi.org/10.17504/protocols.io.be8tjhwn Copy   

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Authors: Shashank Sathe, Clarence Mah, Noorsher Ahmed, Michelle Franc Ragsac, John Williams
Group: Coronavirus Method Development Community
Summary: A specimen to data tracking tool for SEARCH SARS-CoV-2 tests. The application is used by SEARCH technicians to track samples as they proceed through each step within the RT-qPCR testing workflow. The app is currently hosted here: http://inspect-covid.com/qpcr_records/

Proper citation: Shashank Sathe, Clarence Mah, Noorsher Ahmed, Michelle Franc Ragsac, John Williams 2020. INSPECT sample tracking system. protocols.io https://dx.doi.org/10.17504/protocols.io.bis8kehw 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.4 - 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.bpdfmi3n Copy   

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Authors: Chantal Vogels, Joseph Fauver, Isabel Ott, Nathan Grubaugh
Group: Coronavirus Method Development Community
Summary: The protocol describes how to generate high-quality single-stranded RNA transcript standards (starting from a virus RNA stock) targeting the nsp10, RdRp, nsp14, envelope (E), and nucleocapsid (N) coding regions for use with China CDC, Hong Kong University (HKU), Corman et al. (Berlin), and US CDC SARS-CoV-2 primer and probe sets for qRT-PCR. (Sequences for transcripts generated, along with their corresponding assays, are provided under 'Guidelines.')

Proper citation: Chantal Vogels, Joseph Fauver, Isabel Ott, Nathan Grubaugh 2020. Generation of SARS-COV-2 RNA transcript standards for qRT-PCR detection assays. protocols.io https://dx.doi.org/10.17504/protocols.io.bdv6i69e Copy   

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Authors: Valeriia Kaptelova, Speranskaya AS, et.al
Group: Coronavirus Method Development Community
Summary: Here we present a new primer panel that allows amplifying the complete genome of SARS-CoV-2 (the causative virus of COVID-19) using 17 primer pairs (in four pools). Our results demonstrate that our method allows producing full genomes when we use RNA extract from SARS-CoV-2 positive clinical samples which have a cycle threshold (Ct) in the range of 13 to 26. The resulting primer set exhibits the coverage of the entire viral genome except for only 8 bp on 5'- and 80 bp on 3'- ends in comparison with the reference genome in GenBank (accession number MT121215.1).

Proper citation: Valeriia Kaptelova, Speranskaya AS, et.al 2020. Protocol for SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bn77mhrn Copy   

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Authors: Monica Simion, Alexandru Salceanu
Group: Coronavirus Method Development Community, covid19-test, XPRIZE Rapid Covid Testing

Proper citation: Monica Simion, Alexandru Salceanu 2020. microfluidic sars cov 2 lamp protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bkjjkukn 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, Charlotte Beaver, Rachel Nelson, Alex Alderton, Ian Johnston
Group: Coronavirus Method Development Community
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, Charlotte Beaver, Rachel Nelson, 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.be3wjgpe Copy   

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Authors: Shuvam Banerjee, Sohan Seal, Riju Dey, Kousik Kr. Mondal, Pritha Bhattacharjee
Group: Coronavirus Method Development Community
Summary: Pandemic COVID-19 outbreak has been caused due to SARS-COV2 pathogen, resulting millions of infection and death worldwide, USA being on top at the present moment. The long, complex orf1ab polyproteins of SARS-COV2 play an important role in viral RNA synthesis. To assess the impact of mutations in this important domain, we analyzed 1134 complete protein sequences of orf1ab polyprotein from NCBI Virus database from affected patients across various states of USA from December 2019 to 25thApril, 2020. Multiple sequence alignment using Clustal Omega followed by statistical significance was calculated. Four significant mutations T265I (nsp 2), P4715L (nsp 12) and P5828L and Y5865C (both at nsp 13) were identified in important non-structural proteins, which function either as replicase or helicase. A comparative analysis shows 265T>I, 5828P>L and 5865Y>C are unique to USA and not reported from Europe or Asia; while one, 4715P>L is predominant in both Europe and USA. Mutational changes in amino acids are predicted to alter structure and function of corresponding proteins, thereby it is imperative to consider the mutational spectra while designing new antiviral therapeutics targeting viral orf1ab.

Proper citation: Shuvam Banerjee, Sohan Seal, Riju Dey, Kousik Kr. Mondal, Pritha Bhattacharjee 2020. Mutational Spectra of SARS-CoV-2 orf1ab polyprotein and Signature mutations in the United States of America. protocols.io https://dx.doi.org/10.17504/protocols.io.bgesjtee Copy   

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Authors: John Daniels
Group: Coronavirus Method Development Community, Front Line Technologies, XPRIZE Rapid Covid Testing, Team Exhaled Breath Condensate
Summary: The Kinaptic mask-based COVID-19 testing system uses a unique Exhaled Breath Condensate (EBC) collector that converts breath vapor to a liquid biosample for rapid testing and wireless automatic contact tracing. Exhaled Breath Condensate (EBC) is collected from a mask-based EBC collector as a low cost disposable component. Our electronic biosensor detects even low concentrations of virus biomarkers if they are present in the EBC sample. Corona viruses have slowly evolving N-protein and quickly evolving S-protein. N-Protein testing screens for any corona virus infection / S-Protein screens for only COVID-19 infection. The Kinaptic mask-based COVID-19 testing system is a “two-stage filter”: 1) catch only of N-protein biomarker = trigger warning to get more stringent diagnostic and contact trace; 2) catch both N- and S-, or just S- biomarkers = more urgent need for immediate quarantine and medical attention. As the target biomarkers bind to the capture molecules, the electrical conductivity of the electronic biosensor changes. This change in conductive is amplified by our bluetooth electronics, analyzed to determine the presence and quantity of the target biomarker, and the test result wireless transmitted to a smartphone or directly to the cloud.

Proper citation: John Daniels 2020. Mask-Based Covid-10 testing system using Exhaled Breath Condensate. protocols.io https://dx.doi.org/10.17504/protocols.io.bkzzkx76 Copy   

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Authors: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton
Group: Coronavirus Method Development Community
Summary: In the United States, access to testing for the novel coronavirus (SARS-Cov2) is severely limited. Arguably, the PCR based tests are the most reliable when it comes to detecting the virus. Critical reagents for these tests, however, are in short supply. Our group has worked to identify and test alternative reagents and supplies that are not in high demand by clinical labs. We have adapted a more traditional approach to isolating RNA does not use a kit. RNA isolated can be used in traditional quantitative PCR or droplet digital PCR, which has been shown to be ~500 times more sensitive.

Proper citation: Joseph Patterson, Allyson Cole-Strauss, John Beck, Caryl Sortwell, Jack Lipton 2020. Detection of SARS-Cov2 Without High Demand Reagents (Singleplex Assays). protocols.io https://dx.doi.org/10.17504/protocols.io.be8sjhwe Copy   

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Authors: Hannah Greenwald, Lauren Kennedy, Vinson Fan, Rose Kantor, Kara Nelson
Group: Coronavirus Method Development Community, Wastewater-based epidemiology working group, Wastewater SARS-CoV-2
Summary: The following protocol describes the reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods that we have used at the University of California, Berkeley in the Nelson Lab and COVID-WEB (covid-web.org) for downstream analysis of extracted wastewater samples for the purpose of SARS-CoV-2 surveillance. We have been successfully using this protocol paired with the Sewage, Salt, Silica, and SARS-CoV-2 (4S) method. We are including multiple assays that we have used throughout this project, although not all of them may be in active use. This method may need to be adapted based on your lab's application, available equipment, and safety requirements. 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.Version 1 included the following assays: N1, PMMoV, BCoV, and SOC.Version 2 was amended to include the following assays: crAssphage, Bacteroides HF183 rRNA, and human 18S rRNA.

Proper citation: Hannah Greenwald, Lauren Kennedy, Vinson Fan, Rose Kantor, Kara Nelson 2021. One-Step RT-qPCR for SARS-CoV-2 Wastewater Surveillance: N1, PMMoV, BCoV, SOC, CrAssphage, Bacteroides rRNA, 18S rRNA . protocols.io https://dx.doi.org/10.17504/protocols.io.bsgvnbw6 Copy   

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Authors: Max Marrone
Group: Coronavirus Method Development Community, Opentrons COVID-19 Testing

Proper citation: Max Marrone 2020. Operating an OT-2 for COVID-19 testing. protocols.io https://dx.doi.org/10.17504/protocols.io.bejejcje Copy   

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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 9 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. Statistical Considerations (Part 9 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bj55kq86 Copy   

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Authors: Mario Thomas, Jasmine Sollen, Natalia Ivanova, Michelle Feng, Reda Fayek, Heidi Abdilla, Stephanie Lim, Amanda Naaum
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, SANATA
Summary: This procedure outlines the protocol for testing for SARS-CoV-2 using a saliva sample collected from an individual. The purpose of this test is to detect low levels of SARS-CoV-2 antigen at a higher sensitivity. Precision Biomonitoring Inc. developed an ultra-rapid digital, disposable, highly-sensitive and inexpensive testing device used for screening purposes. The mobile app complementary to this medical device is connected through Bluetooth. Using this innovation, the user can be tested at point-of-care (POC) by a health care professional, and obtain qualitative results.

Proper citation: Mario Thomas, Jasmine Sollen, Natalia Ivanova, Michelle Feng, Reda Fayek, Heidi Abdilla, Stephanie Lim, Amanda Naaum 2020. XPRIZE SANATA Protocol for Saliva LFIA Test. protocols.io https://dx.doi.org/10.17504/protocols.io.bqfrmtm6 Copy   

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Authors: Josh Quick
Group: Diaz-Munoz Lab, Coronavirus Method Development Community, Szentagothai Research Centre Virology

Proper citation: Josh Quick 2019. Starting a MinION sequencing run using MinKNOW. protocols.io https://dx.doi.org/10.17504/protocols.io.7q6hmze Copy   

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Authors: vincent.degennaro
Group: Coronavirus Method Development Community, API Pharma LLC, XPRIZE Rapid Covid Testing

Proper citation: vincent.degennaro 2020. API Pharma Next Gen Antibody Test. protocols.io https://dx.doi.org/10.17504/protocols.io.bjqqkmvw Copy   

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Authors: Max Marrone
Group: Coronavirus Method Development Community, Opentrons COVID-19 Testing

Proper citation: Max Marrone 2020. Cleaning an OT-2 COVID-19 Diagnostic Station. protocols.io https://dx.doi.org/10.17504/protocols.io.beb5jaq6 Copy   

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