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Authors: Lusajo Mwakibete, Cristina Tato, Vida Ahyong, Amy Kistler, Manu Vanaerschot, Angela Detweiler, Michelle Tan
Group: Chan Zuckerberg Biohub, Coronavirus Method Development Community

Proper citation: Lusajo Mwakibete, Cristina Tato, Vida Ahyong, Amy Kistler, Manu Vanaerschot, Angela Detweiler, Michelle Tan 2021. ARTIC-NEB: SARS-CoV-2 Library Prep. protocols.io https:// Copy   

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Authors: Ronald W.A.L. Limpens, Eric J. Snijder, Abraham J. Koster, Montserrat Bárcena
Group: Coronavirus Method Development Community
Summary: This protocol allowed the subcellular localisation of viral RNA synthesis in coronavirus-infected cells by EM autoradiography, though it is largely applicable to other types of EM autoradiography samples. Newly synthesized viral RNA is metabolically labelled by providing the infected cells with tritiated uridine while arresting cellular transcription. Subsequently, the samples are processed for EM, sectioned and incubated with a photographic emulsion. Radioactive disintegrations from the labelled viral RNA generate signal around the radioactive source that, after development, becomes apparent as electron-dense grains that can be detected by transmission electron microscopy. The advantages of this approach are its high sensitivity and its compatibility with high-contrast EM sample preparation protocols. The protocol presented here is an adaptation of different methods and tips to EM autoradiography discussed in detail by Williams, in Glauert, 1977, Practical Methods in Electron Microscopy, Volume 6, Chapter 4.

Proper citation: Ronald W.A.L. Limpens, Eric J. Snijder, Abraham J. Koster, Montserrat Bárcena 2020. Subcellular localisation of newly synthesized viral RNA in coronavirus infection by EM autoradiography. protocols.io https://dx.doi.org/10.17504/protocols.io.bfrtjm6n Copy   

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Authors: Camila Romano, Jaqueline De Jesus, Alvina Clara Felix, Anderson V de Paula, Pâmela S Andrade, Franciane M de Oliveira, Darlan Cândido, Nuno Faria, William M de Souza, Ester C Sabino
Group: Coronavirus Method Development Community
Summary: The recently emerged variants of concern (VOC) B.1.1.7 in UK, B.1.1.35 in South Africa and P.1 in Brazil have increased transmissibility and are rapidly spreading worldwide. These variants have several substitutions that allow them to bypass the immune responses and are up to 10 times more contagious than the previous circulating SARS-COV-2 lineages. While in some regions P.1 predominates (as several estates in Brazil), in other countries B.1.1.7 or B.1.1.35 are most prevalent. Therefore, real-time surveillance of VOC is the key for rapid public health responses. In this matter, RT-PCR protocols that are capable of detecting VOCs easily and rapidly are needed. We developed and validated a protocol that detect simultaneously the 3 most widespread VOC. Although this protocol does not discriminate among the VOCs, it can reliably detect mixed infection. Importantly, our protocol successfully identified a co-infection by two SARS-COV-2 lineages (P.1 and B.1), confirmed through complete genome sequencing.

Proper citation: Camila Romano, Jaqueline De Jesus, Alvina Clara Felix, Anderson V de Paula, Pâmela S Andrade, Franciane M de Oliveira, Darlan Cândido, Nuno Faria, William M de Souza, Ester C Sabino 2021. Real-Time PCR protocol to screen for SARS-COV-2 variants of concern (B.1.1.7, P.1 and B.1.1.35) . protocols.io https://dx.doi.org/10.17504/protocols.io.bszbnf2n Copy   

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Authors: Pierre Teodosio Felix, Robson da Silva Ramos, Dallynne Bárbara Ramos Venâncio, Cícero Batista do Nascimento Filho, Antônio João Paulino
Group: Coronavirus Method Development Community, LaBECom
Summary: In this work, we evaluated the levels of genetic diversity in 38 complete genomes of SARS-CoV-2, publicly available on the National Center for Biotechnology Information (NCBI) platform and from six countries in South America (Brazil, Chile, Peru, Colombia, Uruguay and Venezuela with 16, 11, 1, 1, 1, 7 haplotypes, respectively), all with an extension of 29,906 bp and Phred values ≥ 40. These haplotypes were previously used for phylogenetic analyses, following the alignment protocols of the MEGA X software; where all gaps and unconserved sites were extracted for the construction of phylogenetic trees. The specific methodologies for Paired FSTestimators, Molecular Variance (AMOVA), Genetic Distance, mismatch, demographic and spatial expansion analyses, molecular diversity and evolutionary divergence time analyses, were obtained using 20,000 random permutation.

Proper citation: Pierre Teodosio Felix, Robson da Silva Ramos, Dallynne Bárbara Ramos Venâncio, Cícero Batista do Nascimento Filho, Antônio João Paulino 2020. Mathematical models for understanding the genetic diversity of the new coronavirus: LaBECom's analytical protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.bmbvk2n6 Copy   

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Authors: Nicola O'Reilly, Svend Kjaer
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 L6 5M Guanidine thiocyanate virus inactivation buffer for the viral inactivation protocol.

Proper citation: Nicola O'Reilly, Svend Kjaer 2020. Preparation of 5M Guanidine thiocyanate L6 Inactivation Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.bfevjje6 Copy   

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Authors: Prem Premsrirut, Ana Vasileva, Huiting Cheng
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: OverviewAt the start of the Pandemic in the US, New York was hit with more cases of COVID-19 than any single country in the world. Although case numbers are finally at an all-time low, the question of how to safely reopen the economy, school and businesses still remains unclear. Today, SARS-CoV-2 detection via RT-PCR methods remains the gold standard to detect active infection and recommend isolation of individuals in order to prevent further spread. Unfortunately, the cost and bottlenecks created from repeated massive testing efforts have become unsustainable. As a way to address this issue, we have devised a strategy of pooling samples and testing entire populations together as one unit. This cuts the cost of testing dramatically and still enables the detection of a susceptible group that would need to undergo individual testing.To meet the COVID-19 testing demands, we created an organizational-based pooling strategy, whereby organizations can enroll in surveillance testing to monitor groups of people through pooled testing. By using organizational-based pooling, we avoid random pooling of samples. Instead, we pool groups of known contacts from a defined workplace, school or organization where people regularly interact. Therefore, a positive case would likely have an impact on the entire pooled population and the entire pooled population can be treated as an infected cohort until further individual testing, potentially, through a healthcare provider is recommended or offered.Method of Pooled TestingFor pooled surveillance testing to be effective and widely adopted, sample collection methods must also be simplified and non-invasive in order to achieve high compliance and enrollment into a surveillance program. The method of testing must also be highly sensitive to accurately detect one positive case in a large pool of negative samples. For these reasons, we developed a simple saliva collection method with a validated pooled saliva specimens using RT-PCR and an acceptable limit of detection, which will be the most sensitive diagnostic test to date. We established automated methods to pool up to 24 saliva specimens and if a positive case is detected, the pools can be further broken down to pools of 2 specimens. The remaining 2 individuals within a pool will be referred to or, potentially, offered further clinical diagnostic testing by a healthcare provider.

Proper citation: Prem Premsrirut, Ana Vasileva, Huiting Cheng 2020. Mirimus COVID-19 Pool Surveillance RT-PCR Testing. protocols.io https://dx.doi.org/10.17504/protocols.io.bkv3kw8n 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. McGill Nanopore Native Barcoding LibPrep Protocol,10 ng NB. protocols.io https://dx.doi.org/10.17504/protocols.io.bmpsk5ne 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 detection10 copies/μl input Figure 1 | a) SARS-CoV-2 DETECTR has a limit of detection (n=6) of 10 copies per µl input. b) Schematic of DETECTR assay coupled to lateral flow strip. c) Representative lateral flow results for the assay shown for 0 copies per µl and 10 copies per µlAcknowledgements: 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.bfhcjj2w Copy   

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Authors: Adélaïde Roguet, Shuchen Feng
Group: Coronavirus Method Development Community, mclellan lab

Proper citation: Adélaïde Roguet, Shuchen Feng 2021. Prepare bovine coronavirus (BCoV) solution. protocols.io https://dx.doi.org/10.17504/protocols.io.bpg8mjzw Copy   

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Authors: Aziz Sheikh, Sandeep Ramalingam, John Norrie, Emma Ward, Catriona Graham
Group: Coronavirus Method Development Community
Summary: Post-hoc secondary analysis of data from our recent Edinburgh and Lothians Viral Intervention Study (ELVIS) pilot randomised controlled trial (RCT) indicates that hypertonic saline nasal irrigation and gargling (HSNIG) reduced the duration of coronavirus upper respiratory tract infection (URTI) by an average of two-and-a-half days. As such, it may offer a potentially safe, effective and scalable intervention in those with Coronavirus Disease-19 (COVID-19) following infection with the betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).ELVIS was undertaken in 66 adults with an URTI. Results have been reported in detail elsewhere. Briefly, volunteers with URTI were, within 48 hours of symptom onset, randomised to intervention (n=32) or control (n=34) groups. The intervention group made hypertonic saline at home and performed HSNIG as many times as needed (maximum of 12 times/day). Control group participants dealt with their URTI as they normally did. Nose swabs collected at recruitment and first thing in the morning on four consecutive days were sent to the laboratory for testing. Both groups kept a diary (which included the Wisconsin Upper Respiratory Symptom Survey-21 questionnaire) for a maximum of 14 days or until they were well for two consecutive days. Follow-up data were available for 92% of individuals (intervention group: n=30; control group: n=31). HSNIG reduced the duration of URTI by 1.9 days (95% CI = 0.4 to 3.3) (p=0.01), over-the-counter medication use by 36% (p=0.004), transmission within household contacts by 35% (p=0.006) and viral shedding by ≥0.5 log10/day (p=0.04) in the intervention group when compared to controls.We also recently reported that epithelial cells mount an antiviral effect by producing hypochlorous acid (HOCl) from chloride ions. HOCl is the active ingredient in bleach. Epithelial cells have this innate antiviral immune mechanism to clear viral infections. Since bleach is effective against all virus types, we tested to see if a range of DNA, RNA, enveloped and non-enveloped viruses were inhibited in the presence of chloride ions supplied via salt (NaCl). All the viruses we tested were inhibited in the presence of NaCl. The human viruses we tested were: DNA/enveloped: herpes simplex virus; RNA/enveloped: human coronavirus 229E (HCoV-229E), respiratory syncytial virus, influenza A virus; and RNA/non-enveloped: coxsackievirus B3.In COVID-19, high titres of SARS-CoV-2 are detectable in the upper respiratory tract of asymptomatic and symptomatic individuals. The titres are higher in the nose than the throat suggesting measures that control the infection and viral shedding will help reduce transmission. In the context of the COVID-19 pandemic, we have undertaken a post-hoc re-analysis of the ELVIS data with a focus on those infected with coronaviruses. Coronaviruses were the second most common cause of URTI (after rhinoviruses). Fifteen individuals were infected by a coronavirus (intervention group (n=7), control group (n=8)). In the intervention group, four participants were infected by an alphacoronavirus (HCoV 229E=3, HCoV NL63=1) and three by a betacoronavirus (HCoV HKU1=3). In the control group, two were infected by an alphacoronavirus (HCoV NL63=2) and six by a betacoronavirus (HCoV OC43=1, HCoV HKU1=5). An individual in the control group with HCoV HKU1 had dual infection with rhinovirus.The duration of illness was lower in the intervention group compared to the control group in the subset of patients infected with coronavirus (mean days (SD): 5.6 (1.4) vs 8.1 (2.9)). Using a two-sample t-test, this was difference of -2.6 days (95%CI -5.2, 0.05; p=0.054). The difference in the duration of blocked nose was -3.1 days (95%CI -6.0, -0.2; p=0.04), cough -3.3 days (95%CI -5.9, -0.7; p=0.02) and hoarseness of voice -2.9 days (95%CI -5.6, -0.3; p=0.03) in favour of HSNIG.In the absence of a suitable antiviral agent or a vaccine, we need a safe and effective intervention that can be globally implemented. Our in-vitro data gives the evidence that NaCl has an antiviral effect that works across viral types. The findings from this post-hoc analysis of ELVIS need to be interpreted with caution. These data do however suggest that HSNIG may have a role to play in reducing symptoms and duration of illness in COVID-19. Primary research objectiveTo investigate whether the use of Hypertonic Saline Nasal Irrigation and Gargling (HSNIG) performed by adults with symptoms consistent with COVID-19 reduces the duration of symptoms when compared to participants managed using standard care.Secondary research objectiveTo determine the effect of HSNIG on:1. Severity of all symptoms2. Duration and severity of individual symptoms3. Over-the-counter medication use4. Contact with primary care, NHS 24/111 and out-of-hours (OOH) primary care, COVID-19 hubs5. Hospital attendance (i.e. A&E attendance and/or hospital admission) and diagnosis6. Number of household contacts infected7. Side-effect of HSNIG.Trial designELVIS COVID-19 is a pragmatic web-based Bayesian adaptive randomised controlled, parallel group trial of hypertonic saline nasal irrigation and gargling compared to standard care in participants with clinically suspected or confirmed COVID-19 being managed at home. Participants from Scotland will be self-recruiting via web based system which will randomise in a 1:1 ratio to perform HSNIG or not.Eligibility criteriaInclusion: ·Adults (≥18 years) ·Those living within Scotland·Those self-isolating at home within 48 hours of the start of the illness with:(a)Clinical symptoms suggestive of COVID-19 (i.e. those who have at least one of the following symptoms: recent onset of (i) new continuous cough and/or (ii) high temperature) and/or (iii) loss of, or change in, sense of smell or taste (anosmia) OR(b)Those with virologically confirmed SARS-CoV-2 infection and clinical symptoms indicative of COVID-19 (as detailed in (a) above).·Provision of informed consentExclusion: ·Onset of illness >48 hours·People ≤17 years·Inability to consent·Pregnancy·Immunosuppression·Inability to perform HSNIG·Those taking part in another interventional medical trial·Those without access to a supply of salt·Those who have had a negative COVID-19 swab result for the present symptoms ·Those with suspected/confirmed COVID-19 in whom hospital admission is recommended ·Those who do not have access to email/internet·Those living in a household with another person currently participating in this studySettingThis study will look for participants across Scotland, who are 18 years old or older and who are self-isolating with confirmed or suspected COVID-19, with symptoms that have developed no more than 48 hours before consent to the study. The study is conducted online and everything we ask participants to do will be done at home with items they will already have.RandomisationParticipants will be allocated in 1:1 ratio to either HSNIG or standard care.The randomisation will be performed using a web-based system utilising a block randomisation with varying block sizes and will be stratified on the variables: age (InterventionParticipants will be randomised into either the intervention or the control group. The intervention group will be asked to perform HSNIG up to 12 times daily for a maximum of 14 days or until they report that they feel well. The control group will be given standard NHS guidance for the management of their symptoms and household hygiene. Both groups will be sent a daily diary via email to complete online as soon as possible that day. If a participant reports that they are feeling well before the end of the 14 days, they will receive an End of Illness questionnaire to complete, as well as a short questionnaire on Day 14.Primary EndpointSelf-reported time to resolution as assessed by completion of the validated self-reported UK-adapted short form of the Wisconsin Upper Respiratory Symptom Survey (WURSS-24), which will be used to collect daily symptom dataSecondary Endpoints1. Severity of all symptoms2. The length of time for individual symptoms to resolve3. Severity of individual symptoms4. Contacting healthcare (NHS 24, OOH, GP) (Number of participants and frequency of contacts)5. Participants needing GP appointments (Number of participants and frequency of contacts)6. Participants attending hospital (Number of participants)7. Length of stay in hospital if admitted8. Number of participants reporting over the counter medication use9. Reduction in transmission to household contacts10. Number of participants reporting side effects of nasal irrigation11. Types and severity of side effects reported12. Cost of over the counter medication usedSample sizeWe aim to recruit a total of 405 participants from within Scotland. 50% of these patients will be randomised to the study intervention and 50% will be randomised to standard care.

Proper citation: Aziz Sheikh, Sandeep Ramalingam, John Norrie, Emma Ward, Catriona Graham 2020. Hypertonic saline nasal irrigation and gargling for suspected or confirmed COVID-19: pragmatic web-based Bayesian adaptive randomised controlled trial (ELVIS COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bhrnj55e Copy   

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Authors: Katy Graham, Marlene Wolfe, Stephanie Loeb, Alexandria Boehm, Krista Wigginton
Group: Coronavirus Method Development Community, Wastewater-based epidemiology working group
Summary: This SOP describes pre-analytical procedures to be followed for the isolation and identification of SARS-CoV-2 RNA in influent wastewater samples from wastewater treatment plants. PEG precipitation is used to concentrate viruses in the samples. The concentrated pellet is recovered and resuspended to be utilized for the next steps of the protocol. Bovine coronavirus is used as a recovery control for the concentration step. The RNA extraction is acheived using a commercial kit and an inhibitor removal kit is used to remove inhibitors that were co-extracted with the RNA. The RNA from this protocol can be used in droplet digital RT-PCR assays. A protocol for the dd-RTPCR assays is available from our group. Biosafety ConcernsConcentration and extraction procedures that utilize raw samples must adhere to strict Biosafety Level 2+ procedures. These procedures should be performed in a dedicated room. Downstream products may be handled using standard laboratory safety guidelines.

Proper citation: Katy Graham, Marlene Wolfe, Stephanie Loeb, Alexandria Boehm, Krista Wigginton 2020. Concentration of viruses from wastewater influent using organic flocculation (PEG). protocols.io https://dx.doi.org/10.17504/protocols.io.bi6jkhcn Copy   

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Authors: Marie-Michelle Simon, Sarah Reiling, Ioannis Ragoussis
Group: Coronavirus Method Development Community, McGill Genome Centre
Summary: How the Nextera DNA Flex Assay Works The Nextera DNA Flex library prep kit uses a bead-based transposome complex to tagment genomic DNA, which is a process that fragments DNA and then tags the DNA with adapter sequences in one step. After it is saturated with input DNA, the bead-based transposome complex fragments a set number of DNA molecules. This fragmentation provides flexibility to use a wide DNA input range to generate normalized libraries of consistent tight fragment size distribution. Following tagmentation, a limited-cycle PCR adds Nextera DNA Flex-specific index adapter sequences to the ends of a DNA fragment. This step enables capability across all Illumina sequencing platforms. A subsequent Sample Purification Beads (SPB) cleanup step then purifies libraries for use on an Illumina sequencer. The double-stranded DNA library is denatured before hybridization of the biotin probe oligonucleotide pool. PCR Amplicons for Nextera FlexWhen starting with PCR amplicons, the PCR amplicon must be > 150 bp. The standard clean up protocol depletes libraries Therefore, Illumina recommends that amplicons Shorter amplicons can otherwise be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end. More information can be found here: https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdf

Proper citation: Marie-Michelle Simon, Sarah Reiling, Ioannis Ragoussis 2020. nCoV-2019 McGill Nextera Flex LibPrep Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bjgnkjve Copy   

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Authors: Erica Wells
Group: Coronavirus Method Development Community
Summary: Our limited findings: This “DIY folding mask” tries to replicate some of the benefits of a n95 mask. It has been saccharin tested by several nurses and an NP. I cannot say these are as effective as a factory n95 mask because of course a real one would be better. But I tested it against a CDC recommended bandana and scarf and it seems to out perform them. It also seems to outperform the surgical mask.

Proper citation: Erica Wells 2020. Mask Building Idea. protocols.io https://dx.doi.org/10.17504/protocols.io.bd3ji8kn Copy   

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Authors: Jennifer Giandhari, Sureshnee Pillay, Houriiyah Tegally, Eduan Wilkinson, Benjamin Chimukangara, Richard Lessells, Yunus Moosa, Inbal Gazy, Maryam Fish, Lavanya Singh, Khulekani Sedwell Khanyile, Vagner Fonseca, Marta Giovanetti, Luiz Carols Alcantara, Tulio de Oliveira
Group: Coronavirus Method Development Community, KRISP
Summary: This protocol describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing amplicons tiling the viral genome sequencing. It uses the V3 nCov-2019 primers from the ARTIC network. This is followed by library construction using Nextera Flex, which we found to save 9h of hands on time as compared with original protocol that uses TruSeq for library contstruction. It also describes the pooling of samples and quantitation, prior to sequencing on the Illumina Miseq. It is adapted from the nCov-2019 sequencing protocol from Quick and colleagues, which can be found here:

Proper citation: Jennifer Giandhari, Sureshnee Pillay, Houriiyah Tegally, Eduan Wilkinson, Benjamin Chimukangara, Richard Lessells, Yunus Moosa, Inbal Gazy, Maryam Fish, Lavanya Singh, Khulekani Sedwell Khanyile, Vagner Fonseca, Marta Giovanetti, Luiz Carols Alcantara, Tulio de Oliveira 2020. NEBnext library construction and sequencing for SARS-CoV-2: Adapting COVID-19 ARTIC protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bhu2j6ye 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 2021. Protocol for SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing.v3.Neb. protocols.io https://dx.doi.org/10.17504/protocols.io.bsybnfsn 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 dual-plex 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)

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.bhfej3je Copy   

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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.br9vm966 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.

Proper citation: Hannah Greenwald, Lauren Kennedy, Vinson Fan, Rose Kantor, Kara Nelson 2020. One-Step RT-qPCR for SARS-CoV-2 Wastewater Surveillance: N1, PMMoV, BCoV, SOC . protocols.io https://dx.doi.org/10.17504/protocols.io.bpk3mkyn 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. UPDATE: The Rapid barcoding protocol is now (March 2021) available on the Oxford Nanopore website for 1-96 samples "PCR tiling of SARS-CoV-2 virus with rapid barcoding (SQK-RBK110.96)" https://community.nanoporetech.com/protocols/pcr-tiling-of-sars-cov-2-virus-with-rapid-barcoding-sqk-rbk110/v/PCTR_9125_v110_revA_24Mar2021This is a modification of the ARTIC amplicon V3 sequencing protocol for MinION for nCoV-2019 developed by Josh Quick. The ARTIC method uses 400 base pair amplicons and the Oxford Nanopore Ligation barcoding kit (LSK-109). The protocol described here, dubbed the Midnight protocol, makes two important changes to the ARTIC V3 protocol: We have increased the size of the amplicon size to 1200bp. Using just 29 amplicons, the midnight primers anneal to only 4.5% of the viral genome compared to 17% or more of the genome, lowering the chance that viral mutations will cause dropouts due to primer mismatches. We use the RAPID barcode kit RBK004 (or, available as of March 2021 for 1-96 samples with the RBK110-96), which requires less time and fewer reagents than the ligation based LSK-109 protocol.The amplicons produced in this protocol can also be used for Illumina sequencing (i.e. using a transposon based shearing like Nextera or the Lotus kit https://sg.idtdna.com/pages/landing/coronavirus-research-reagents/ngs-assays#resources)The original publication that describes this protocol is here along with important considerations is here: https://academic.oup.com/biomethods/article/5/1/bpaa014/5873518?login=truePrimers were all designed using Primal Scheme: http://primal.zibraproject.org/, described here https://www.nature.com/articles/nprot.2017.066.Primer sequences are here (and listed directly in the protocol) 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: V5: Changed Reverse transcription part of the protocol to use the LunaScript™ RT SuperMix Kit in place of SSIV as it is easier, performs similarly, and is less expensive. The PCR step was changed to use the Q5®Hot Start High-Fidelity 2X Master Mix in place of the Q5 reagents individually, as this cuts down pipetting steps, performs similarly, and is similar in cost.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 2021. SARS-CoV2 genome sequencing protocol (1200bp amplicon "midnight" primer set, using Nanopore Rapid kit). protocols.io https://dx.doi.org/10.17504/protocols.io.btsrnnd6 Copy   

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Authors: Ying Tang, Carly G.K. Ziegler, Vincent N. Miao, Andrew W. Navia, Joshua D. Bromley, Kenneth J. Wilson, Yilianys Pride, Mohammad Hasan, Taylor Christian, Hannah Laird, Anna Owings, Meredith Sloan, Haley B. Williams, Tanya O. Robinson, George E. Abraham III, Michal Senitko, Sarah C. Glover, Bruce Horwitz, Alex K. Shalek, Jose Ordovas-Montanes
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: A protocol for recovering viable single cell suspensions from cryopreserved human nasopharyngeal swabs for downstream applications, such as single-cell RNA-seq. The illustrated schematic below details the process.

Proper citation: Ying Tang, Carly G.K. Ziegler, Vincent N. Miao, Andrew W. Navia, Joshua D. Bromley, Kenneth J. Wilson, Yilianys Pride, Mohammad Hasan, Taylor Christian, Hannah Laird, Anna Owings, Meredith Sloan, Haley B. Williams, Tanya O. Robinson, George E. Abraham III, Michal Senitko, Sarah C. Glover, Bruce Horwitz, Alex K. Shalek, Jose Ordovas-Montanes 2020. Human Nasopharyngeal Swab Processing for Viable Single-Cell Suspension. protocols.io https://dx.doi.org/10.17504/protocols.io.bjhkkj4w Copy   

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