Protocols   Select Another Resource Report Type

Authors: Nikki Freed, Olin Silander
Group: Coronavirus Method Development Community
Summary: Instructions to quantify double stranded DNA on a Qubit Fluorometer using the high sensitivity dsDNA assay kit

Proper citation: Nikki Freed, Olin Silander 2020. DNA quantification using the Qubit fluorometer. protocols.io https://dx.doi.org/10.17504/protocols.io.bfy3jpyn Copy   

•   Source:

Authors: Paola Resende
Group: Coronavirus Method Development Community
Summary: This protocol describes step-by-step instructions for building long (~2kb) amplicon libraries to recover SARS-CoV-2 genomes using the nanopore sequencing.It can be applied for sequencing on the MinION device or GridION. This protocol was developed in collaboration with the Laboratory of Respiratory Viruses and Measles, Oswaldo Cruz Institute, FIOCRUZ, Brazil, and the two sequencing facilities at Pathogen Genomic Unit (PGU) and UCL Genomics, University College London (UCL), United Kingdom. This document describes the manual steps to perform the protocol, but for further information about the automation of the protocol, please, contact Pathogen Genomics Unit (PGU) - UCL. Manager: Rachel Williams https://www.ucl.ac.uk/infection-immunity/pathogen-genomics-unitThis protocol is based on the amplicon tiling strategy described previously by Quick J et al 2017. However, we have applied this strategy to recover long reads (2kb), then some adjustments were performed in the protocol.If you have doubts, we are happy to help you to test or implement the protocol.

Proper citation: Paola Resende 2020. Long reads nanopore sequencing to recover SARS-CoV-2 whole genome . protocols.io https://dx.doi.org/10.17504/protocols.io.bfy7jpzn Copy   

•   Source:

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 https://dx.doi.org/10.17504/protocols.io.biwfkfbn Copy   

•   Source:

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 https://dx.doi.org/10.17504/protocols.io.bqnqmvdw Copy   

•   Source:

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 https://dx.doi.org/10.17504/protocols.io.bisbkean Copy   

•   Source:

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.bge5jtg6 Copy   

•   Source:

Authors: Qianxin Wu, Chenqu Suo, Tom Brown, Tengyao Wang, Sarah Teichmann, Andrew Bassett
Group: Coronavirus Method Development Community
Summary: We present here INSIGHT (Isothermal NASBA-Sequencing based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a combination of an isothermal NASBA reaction and next generation sequencing. From commercially acquired human saliva with spiked-in viral RNA as input, the first stage employs isothermal amplification of viral RNA to give a rapid result in one to two hours, using either fluorescence detection or a dipstick readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. In the first stage, fluorescent viral RNA detection can be consistently achieved at 10-100 copies per 20 μl reaction. The second stage pools post-amplification barcoded products from multiple samples for scalable sequencing that could be centralised, to further improve the accuracy of the test in a massively parallel way. Our two-stage testing strategy is suitable for further development into a home-based or point-of-care assay, and is potentially scalable to population level.

Proper citation: Qianxin Wu, Chenqu Suo, Tom Brown, Tengyao Wang, Sarah Teichmann, Andrew Bassett 2020. INSIGHT: a scalable isothermal NASBA-based platform for COVID-19 diagnosis. protocols.io https://dx.doi.org/10.17504/protocols.io.bghsjt6e Copy   

•   Source:

Authors: Sarah Reiling, Josh Quick, Ioannis Ragoussis
Group: Coronavirus Method Development Community, McGill Genome Centre
Summary: V3 only primers for this protocol were designed using Primal Scheme and generate overlapping 400 nt amplicons. Primer names and dilutions are listed in the table below. https://github.com/sarahreiling/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019_V3only.scheme.bed

Proper citation: Sarah Reiling, Josh Quick, Ioannis Ragoussis 2020. SARS-CoV-2 McGill Artic PCR Protocol, 2.5 ul RT and V3 only + LA1. protocols.io https://dx.doi.org/10.17504/protocols.io.bjgkkjuw Copy   

•   Source:

Authors: Timothy Hamerly, Caroline J Stephenson, Borja Lopez-Gutierrez, Rhoel Dinglasan
Group: Coronavirus Method Development Community, Dinglasan Laboratory
Summary: Diagnostic testing for SARS-CoV-2 by RT-qPCR has steadily increased since the COVID-19 pandemic began, resulting in the development of protocols for testing from a variety of biological matrices. Initial testing focused on nasopharyngeal and oropharyngeal swabs, which are invasive and require trained health care professionals to perform the sample collection, resulting in a bottleneck in testing. Saliva has emerged as a viable sample specimen for virus detection and can be self-collected with little instruction. Further, it has been demonstrated that virus detection is possible without the need for complex extraction protocols, including commercially available kits. However, a potential pitfall of available kits is the lack of comprehensive testing during development using contrived biological samples containing intact, heat inactivated SARS-CoV-2 virus in the appropriate matrix (e.g., saliva). We have found the Takara Direct One-Step RT-qPCR Mix for SARS-CoV-2 kit to be a cost-effective alternative for testing the direct detection of virus in saliva when compared with magnetic bead-assisted extraction systems, with minimal modifications from the manufacturers protocol. We have found that successful detection of virus requires the addition of a proteinase K digestion step, an increase of the reverse transcriptase time, and 3-step cycle (compared with a 2-step cycle in the manufacturers documentation). These changes allow for the successful detection of SARS-CoV-2 virus with a minimal increase in Ct values for RNase P and viral N-gene transcripts when compared with magnetic bead-assisted extractions kits. Implementing these critical steps will help end-users avoid false negatives.

Proper citation: Timothy Hamerly, Caroline J Stephenson, Borja Lopez-Gutierrez, Rhoel Dinglasan 2021. SARS Direct Saliva Protocol Takarabio. protocols.io https://dx.doi.org/10.17504/protocols.io.btzsnp6e Copy   

•   Source:

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 https://dx.doi.org/10.17504/protocols.io.betrjem6 Copy   

•   Source:

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

Proper citation: Adélaïde Roguet, Shuchen Feng 2020. Quantification by Droplet Digital PCR (ddPCR). protocols.io https:// Copy   

•   Source:

Authors: steve.puts
Group: Coronavirus Method Development Community, Mmolecular, XPRIZE Rapid Covid Testing
Summary: The Direct One-Step RT-qPCR COVID-19 Test Kit is designed for quantitativereal-time analysis of target RNA directly from, swabs without the requirement of any priorRNA purification steps.Shipping: shipped on blue ice Storage Conditions: store at -20 °C Additional Storage Conditions: avoid freeze/thaw cycles stable at 4 °C for up to 4 weeks Shelf Life: 12 monthsForm: liquid / lyophilized

Proper citation: steve.puts 2020. Mmolecular COVID-19 Extraction-Free Direct-One-Step Fast Cycling Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bkjpkumn Copy   

•   Source:

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 https://dx.doi.org/10.17504/protocols.io.bs8bnhsn Copy   

•   Source:

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 https://dx.doi.org/10.17504/protocols.io.bhk4j4yw Copy   

•   Source:

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

Proper citation: Max Marrone 2020. Opentrons COVID-19 testing: Station B, Zymo kit, 24 samples. protocols.io https://dx.doi.org/10.17504/protocols.io.be47jgzn Copy   

•   Source:

Authors: Nicola O'Reilly
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 0.1M Tris HCl pH 6.4 for making L6 virus inactivation buffer.

Proper citation: Nicola O'Reilly 2020. Preparation of 0.1M Tris HCl, pH 6.4. protocols.io https://dx.doi.org/10.17504/protocols.io.bfeyjjfw Copy   

•   Source:

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 https://dx.doi.org/10.17504/protocols.io.7q5hmy6 Copy   

•   Source:

Authors: Public Health Ontario
Group: Coronavirus Method Development Community

Proper citation: Public Health Ontario 2020. SARS-CoV-2 Illumina MiSeq protocol v.1. protocols.io https:// Copy   

•   Source:

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

Proper citation: Adélaïde Roguet, Shuchen Feng 2020. Nucleic acid extraction - RNeasy PowerMicrobiome Kit (Qiagen). protocols.io https:// Copy   

•   Source:

Authors: Nicola O'Reilly
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 5M NaOH for making L6 virus inactivation buffer.

Proper citation: Nicola O'Reilly 2020. Preparation of 5M NaOH. protocols.io https://dx.doi.org/10.17504/protocols.io.bfewjjfe Copy   

•   Source: