Protocols   Select Another Resource Report Type

Authors: Bei Wang, Daniel Asarnow, Wen-Hsin Lee, Ching-Wen Huang, Bryan Faust, Patricia Miang Lon Ng, Eve Zi Xian Ngoh, Markus Bohn, David Bulkley, Andrés Pizzorno, Hwee Ching Tan, Chia-Yin Lee, Rabiatul Adawiyah Minhat, Olivier Terrier, Mun Kuen Soh, Frannie Jiuyi Teo, Yvonne Yee Chin Yeap, Yuanyu Hu, Shirley Gek Kheng Seah, Sebastian Maurer-Stroh, Laurent Renia, Brendon John Hanson, Manuel Rosa-Calatrava, Aashish Manglik, Yifan Cheng, Charles S. Craik, Cheng-I Wang
Group: Coronavirus Method Development Community
Summary: In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the “up” and the other in the “down” position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

Proper citation: Bei Wang, Daniel Asarnow, Wen-Hsin Lee, Ching-Wen Huang, Bryan Faust, Patricia Miang Lon Ng, Eve Zi Xian Ngoh, Markus Bohn, David Bulkley, Andrés Pizzorno, Hwee Ching Tan, Chia-Yin Lee, Rabiatul Adawiyah Minhat, Olivier Terrier, Mun Kuen Soh, Frannie Jiuyi Teo, Yvonne Yee Chin Yeap, Yuanyu Hu, Shirley Gek Kheng Seah, Sebastian Maurer-Stroh, Laurent Renia, Brendon John Hanson, Manuel Rosa-Calatrava, Aashish Manglik, Yifan Cheng, Charles S. Craik, Cheng-I Wang 2020. Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization. protocols.io dx.doi.org/10.17504/protocols.io.bi35kgq6 Copy   

•   Source: Protocols.io

Authors: David Eccles
Group: Coronavirus Method Development Community
Summary: This is a fast "gunk to graph" protocol for analysing viral RNA from nasopharyngeal swabs. The approach involves swab lysis and inactivation at the point of sampling, uses a cellulose binding / wash protocol to reduce extraction cost, incorporates sample-specific barcodes during first-strand synthesis, nanopore rapid-attachment primers during PCR amplification, and nanopore sequencing with parallel RAMPART analysis for fast assembly and phylogenetics.Note: this is a one-step RT-PCR reaction protocol. For an alternative method that allows more multiplexing flexibility, see the protocol that this has been forked from:https://dx.doi.org/10.17504/protocols.io.bd3yi8pw

Proper citation: David Eccles 2020. Viral Sequencing, from Gunk to Graph (One-Step four-primer PCR alternative). protocols.io dx.doi.org/10.17504/protocols.io.bd5ni85e Copy   

•   Source: Protocols.io

Authors: Leigh Monahan, Kay Anantanawat, Joyce To, Aaron Darling
Group: Coronavirus Method Development Community
Summary: Managing the current COVID-19 pandemic requires diagnostic testing at an unprecedented scale. However, the crisis has revealed severe deficiencies in our capacity to perform such testing. Here we build on the work of others (1-3) to develop a protocol that not only enables many thousand diagnostic tests to be run in parallel, but also provides near-whole genome sequencing data to facilitate phylogenetic analysis and contact tracing. One of the key features of our protocol is a magnetic bead-based strategy for RNA capture that may circumvent the need for SARS-CoV-2 RNA extraction, currently one of the major bottlenecks in both reagent supply and hands-on sample processing time. This approach also eliminates the requirement for per-sample reverse transcription, significantly reducing per-sample costs. The major steps in our wet lab workflow can be summarised as follows. First, we generate a collection of bead-bound, single-stranded DNA probes tiling the entire SARS-CoV-2 genome. Multiple uniquely barcoded probe sets are prepared, each of which are used to capture viral RNA directly from patient swab samples via DNA/RNA hybridisation. Samples are combined together at this stage, enabling cDNA synthesis to be performed in a single pooled reaction. Finally, multiplex PCR is used to generate a library of overlapping amplicons ready for Illumina sequencing. A schematic overview of the workflow is attached below, along with a more detailed figure depicting the various stages of library preparation.Referenceshttps://docs.google.com/document/d/1kP2w_uTMSep2UxTCOnUhh1TMCjWvHEY0sUUpkJHPYV4/previewhttps://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w/abstracthttps://www.biorxiv.org/content/10.1101/2020.03.20.001008v1.full.pdf

Proper citation: Leigh Monahan, Kay Anantanawat, Joyce To, Aaron Darling 2020. A protocol for massively parallel diagnosis and genome sequencing of SARS-CoV-2. protocols.io dx.doi.org/10.17504/protocols.io.betrjem6 Copy   

•   Source: Protocols.io

Authors: Peng Xu, Venice Servellita, Krzysztof Langer, Dan Weisgerber, Gordon Murtaugh, Adam R Abate, Charles Chiu
Group: Coronavirus Method Development Community, UCSF
Summary: We have developed a highly sensitive and specific LAMP(loop-mediated amplification)-based assay for thedetection of SARS-CoV-2 that can potentially be used for at-home and point-of-care (POC) testing. The assayincludes only three simple steps: (1) heat inactivate the sample mixed with lysis buffer and add LAMP reagentmix, (2) incubate the reaction on a heating device, and (3) read the result by inserting a lateral flow strip intothe tube. Performance is comparable to gold-standard RT-PCR testing, as the assay is based on nucleic aciddetection rather than antigen detection which is used by the majority of lateral flow strip-based assays. There isminimal pre-processing involved and no bulky instrumentation needed, and non-lab professionals can followthe instructions and understand the results easily. The assay targets the SARS-CoV-2 nucleoprotein (N) geneand is capable of detecting as low as 4.0 copies per reaction (0.5 copies/μL) of SARS-CoV-2 RNA,demonstrating no cross-reactivity with a panel of 20 other respiratory pathogens. Furthermore, the test can berun on multiple sample types, including nasopharyngeal and/or oropharyngeal swab and saliva samples.Overall, this assay provides a rapid (sample-to-answer turnaround time of ~40 min), accurate, and affordablesolution to the challenge of at-home diagnostics for SARS-CoV-2.

Proper citation: Peng Xu, Venice Servellita, Krzysztof Langer, Dan Weisgerber, Gordon Murtaugh, Adam R Abate, Charles Chiu 2021. A lateral flow-based at-home test for detection of SARS-CoV-2. protocols.io dx.doi.org/10.17504/protocols.io.bs8bnhsn Copy   

•   Source: Protocols.io

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. In this Version V3, the reverse-transcription step was changed to use of NEB's LunaScript RT SuperMix Kit instead of Thermo Fisher's SuperScript IV Reverse Transcriptase as adapting to the recent update of ARTIC Network's protocol (LoCost). Tyson et al., Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore bioRxiv2020.09.04.283077;doi:https://doi.org/10.1101/2020.09.04.283077 Because the PCR products are fragmented before ligated with adapters, this protocol is compatible to many sequencing kits with various read lengths (75PE, 150PE, etc.) in Illumina machines. While the library preparation uses QiaSeq FX by Qiagen and is basically straight forward (as par kit instruction but modified to 1/4 scale), some tweaks for much of simplicity and speed were added.Change histories (V2): The amount of cDNA input to multiplex PCR is increased (now almost same amount to the ARTIC Network'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).Change histories (V3): RT step has been changed to use of LunaScript (Tyson et al., 2020).Corrected typos and wrong descriptions about amount of reagents.Added a link to library quantification protocol.Other minor changes.Change histories (V3): RT step has been changed to use of LunaScript (Tyson et al., 2020).Corrected typos and wrong descriptions about amount of reagents.Added a link to library quantification protocol.Other minor changes.

Proper citation: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda 2020. nCoV-2019 sequencing protocol for illumina. protocols.io dx.doi.org/10.17504/protocols.io.bnn7mdhn Copy   

•   Source: Protocols.io

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

Proper citation: Josh Quick 2019. Priming and loading a MinION flowcell. protocols.io dx.doi.org/10.17504/protocols.io.7q5hmy6 Copy   

•   Source: Protocols.io

Authors: Gage Moreno, David O'connor
Group: Coronavirus Method Development Community
Summary: This protocol outlines the methods to perform unbiased direct metagenomic sequencing of nucleic acid extracts from cell-free fluids. This protocol can be adapted to be run on Illumina and Nanopore sequencing platforms. The protocol is based off of the work from Kafetzopoulou et al. (PMID: 30563591). Liana has provided the lab with detailed protocols, and has worked with us extensively on optimizing and getting protocols running efficiently. Please note that this protocol has been updated to use SuperScript IV with it’s optimal temperature which has been reflected in Lewandowski et al. (DOI: https://doi.org/10.1128/JCM.00963-19)Notes:* This protocol has been used to sequence influenza direction from respiratory clinical samples (DOI: https://doi.org/10.1128/JCM.00963-19).* A team from China published last week on a 2019-nCoV familial cluster using the SISPA protocol as for coronavirus whole genome sequencing (DOI: https://doi.org/10.1016/S0140-6736(20)30154-9)SISPA-Primer A - 5'-GTT TCC CAC TGG AGG ATA-(N9)-3'SISPA-Primer B - 5′-GTT TCC CAC TGG AGG ATA-3′

Proper citation: Gage Moreno, David O'connor 2020. Sequence-Independent, Single-Primer Amplification of RNA viruses. protocols.io dx.doi.org/10.17504/protocols.io.bhk4j4yw Copy   

•   Source: Protocols.io

Authors: Katharine H. D. Crawford, Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Jesse D. Bloom
Group: Coronavirus Method Development Community
Summary: SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

Proper citation: Katharine H. D. Crawford, Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Jesse D. Bloom 2020. Pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays. protocols.io dx.doi.org/10.17504/protocols.io.bfghjjt6 Copy   

•   Source: Protocols.io

Authors: Justin Ryan, Nicole Coufal, Sage Aronson, Kelsey Ladt, Catelyn Andersen, Mark Zeller, Stephen Rawlings, Denise Malicki, Gene Yeo
Group: Coronavirus Method Development Community
Summary: The global pandemic due to SARS-CoV2 virus, also known as COVID-19, has drastically increased the need for nasopharyngeal-swab-based testing resulting in shortages of commercially available nasopharyngeal (NP) swabs. One solution to overcome the national deficit of swabs is for medical device manufacturers and hospitals to generate NP swabs. Numerous entities are attempting to manufacture a direct from 3D printing NP swab but presented here is a validated two-part swab manufacturing protocol utilizing 3D printing and manual intervention (wrapping of nylon fibers). We recommend material extrusion or powder bed fusion technologies utilizing materials that can be sterilized using high level heat decontamination. Through the application of 3D printing and manual fabrication, we present an NP swab that can be created in a controlled environment. Coupled with CDC published viral transport media, the following swab can be used for COVID-19 testing or for testing for other respiratory viruses (eg, influenza, respiratory syncytial virus). The CDC viral transport medium has only four reagents which are readily available. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Justin Ryan, Nicole Coufal, Sage Aronson, Kelsey Ladt, Catelyn Andersen, Mark Zeller, Stephen Rawlings, Denise Malicki, Gene Yeo 2020. 3D Printed Nasopharyngeal Swabs with Wrapped Rayon Fibers Developed and validated by SCREEN (San Diego Covid19 Research Enterprise Network). protocols.io dx.doi.org/10.17504/protocols.io.bemxjc7n Copy   

•   Source: Protocols.io

Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community
Summary: This is Part 4 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 Population (Part 4 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io dx.doi.org/10.17504/protocols.io.bj5xkq7n Copy   

•   Source: Protocols.io

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: The following protocol describes the "4S" (Sewage, Salt, Silica and SARS-CoV-2) workflow applied to using dry silica powder as an RNA-binding matrix instead of silica spin columns. This offers an even more economical alternative, requiring only centrifugation to extract RNA from wastewater. 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. Direct wastewater RNA extraction via the "Milk of Silica (MoS)" method - A companion method to "Sewage, Salt, Silica and SARS-CoV-2 (4S)". protocols.io dx.doi.org/10.17504/protocols.io.biwfkfbn Copy   

•   Source: Protocols.io

Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community
Summary: This is a collection of protocols for: "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of Vaccine Candidate against COVID-19 in adults ≥ 18 years of age"This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.Please note that this is an evolving document, to be versioned and updated, based on community feedback and new data.

Proper citation: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores 2020. Background and Rationale (Part 1 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io dx.doi.org/10.17504/protocols.io.bj5tkq6n Copy   

•   Source: Protocols.io

Authors: Yang Sun, David Lee, George Hartoularos, Jimmie Ye
Group: Coronavirus Method Development Community
Summary: PurposeTo detail the workflow for scCITE-Seq of 24 PBMC samples with 2 technicians. This is based on internal 10X CITEseq protocol v9.

Proper citation: Yang Sun, David Lee, George Hartoularos, Jimmie Ye 2021. 10X-CITEseq protocol (COVID-19 patient samples +/- tetramer stain). protocols.io dx.doi.org/10.17504/protocols.io.bqnqmvdw Copy   

•   Source: Protocols.io

Authors: Sarah Reiling, Marie-Michelle Simon, Anne-Marie Roy, Shu-Huang Chen, Josh Quick, 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: Sarah Reiling, Marie-Michelle Simon, Anne-Marie Roy, Shu-Huang Chen, Josh Quick, Ioannis Ragoussis 2020. SARS-CoV-2 McGill Nextera Flex sequencing protocol_SS_V3_LA1_5uLRT. protocols.io dx.doi.org/10.17504/protocols.io.bisbkean Copy   

•   Source: Protocols.io

Authors: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
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.

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.bjswknfe Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.bebkjakw Copy   

•   Source: Protocols.io

Authors: Leonardo Caserta
Group: Coronavirus Method Development Community, Diel Lab

Proper citation: Leonardo Caserta 2021. First-strand synthesis and touchdown PCR for SARS-CoV-2. protocols.io dx.doi.org/10.17504/protocols.io.br54m88w Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.7pzhmp6 Copy   

•   Source: Protocols.io

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 dx.doi.org/10.17504/protocols.io.betejeje Copy   

•   Source: Protocols.io

Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: A real-time RT-PCR to designed to detect SARS-CoV-2 and other related sarbecoviruses. Based on sequence MN908947 made available by Professor Yong-Zhen Zhang, Fudan University, Shanghai, China. The target region encodes the nucleocapsid (N).Tested on wild-type SARS-CoV-2 virus , it is expected to be capable of detecting SARS-CoV-2, bat-like SARS and SARS virus (members of the subgenus Sarbecovirus).Limit of detection not yet determined.The performance of the assay has not been tested with low viral load samples or samples from patients who are clinically well.The sensitivity of this assay was improved with the use of the SensiFast™Probe Lo-ROX One-step kit. A single 1 mismatch at probe-binding site identified with the BetaCoV/USA/CA1/2020|EPI_ISL_406034 (GenBank MN994467.1) variant of SARS-CoV-2 (as of 23JUNE2020).Probe is in the 3'-5' (reverse complement) direction.Reverse primers were replaced in March 2020.We also recommend the ORF1ab assay (Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR ORF1ab 2020), US-CDC-N1 assay or the E gene assay by Corman et al. (Protocol v2-1)Notes: Assay is optimised.This test has identified clinical positive cases of coronavirus disease (COVID-19)Notes: Assay is optimised.This test has identified clinical positive cases of coronavirus disease (COVID-19)

Proper citation: Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR N gene 2020 . protocols.io dx.doi.org/10.17504/protocols.io.bhpwj5pe Copy   

•   Source: Protocols.io