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Authors: Monica Galiano, Shahjahan Miah, Angie Lackenby, Omolola Akinbami, Joanna Ellis, Maria Zambon
Group: Coronavirus Method Development Community
Summary: SUMMARYThis document describes the procedure for performing RTPCR amplification for next-generation sequencing (NGS) of whole genomes from SARS-CoV2 positive clinical samples. This methodology was employed at the Respiratory Virus Unit, Microbiology Services Colindale, Public Health England (RVU-PHE) to sequence the first SARS-CoV2 positive samples.Reverse transcription (RT) is performed using random hexamer primers. PCR amplification is done using sequence-specific primers which amplify two sets of 30-31 overlapping amplicons (size 1.0 to 1.8 kb), each set independently covering the entire length of the MERS-CoV genome. Once amplicons are obtained, rough equimolar mixtures, clean-up, DNA quantitation and final dilutions are performed to prepare samples for library preparation for Illumina MiSeq next-generation sequencing (NGS). Primers from set A do not overlap location of primers from set B, except for those binding at the end of the genome (see figure 1). Amplicon mixtures from each set are treated as separate samples throughout the procedure, including assembly. Merging (or comparison) of consensus sequences of both sets is performed at the end. IMPORTANT: Please note that every amplicon is obtained in a separate PCR reaction and, if using both primer sets, there is a total of ~61 PCR reactions; this protocol is NOT a multiplex approach. We recommend using a 96-well plate per sample.ADVANTAGES AND LIMITATIONSThe use of both primer sets allows for redundancy in whole genome (WG) coverage, which increases the chances of obtaining full genome sequences when samples have low viral load. Although we haven’t formally determined the sensitivity of this assay (or limit of amplification of WG), we have obtained full coverage with samples with CT values of up to 38 (CT value obtained at RVU-PHE with RdRp gene detection within the SARS-CoV2 detection protocol as described by Corman et al. [1]). Variation in storage or transport conditions of samples may affect the integrity of viral nucleic acids.Another advantage of this protocol is that, as primers from set A and B match different locations on the genome, direct comparison of the sequences obtained with each set allows for curation of primer-induced contamination of the sequences, although we deal with this with a specific step on the bioinformatic assembly pipeline (not described here).Alternatively, when samples have a relatively good CT (The main limitation of this protocol is its unsuitability for high throughput processing of large batches of samples, although current efforts are being made to adapt the primers to a multiplex approach.
Proper citation: Monica Galiano, Shahjahan Miah, Angie Lackenby, Omolola Akinbami, Joanna Ellis, Maria Zambon 2020. RTPCR Amplification of SARS-CoV2 Whole Genome for Illumina NGS . protocols.io dx.doi.org/10.17504/protocols.io.bew8jfhw Copy
Authors: Judy Northill, Ian Mackay
Group: protocols.io Ambassadors, Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: NOT RECOMMENDED FOR SCREENINGThe sensitivity of the assay has been found to be lower than expected and we no longer recommend it be used.We do recommend the ORF1ab assay (Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR ORF1ab 2020) or the E gene assay by Corman et al. (Protocol v2-1)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).Not tested on wild-type virus (as of 25Jan2020), it is expected to be capable of detecting Wuhan virus, bat-like SARS and SARS virus (members of the subgenus Sarbecovirus).Limit of detection not yet determined.A single 1 mismatch at probe-binding site identified with the BetaCoV/USA/CA1/2020|EPI_ISL_406034 variant of SARS-CoV-2 (as of 29JAN2020).Probe is in the 3'-5' (reverse complement) direction.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).Not tested on wild-type virus (as of 25Jan2020), it is expected to be capable of detecting Wuhan virus, bat-like SARS and SARS virus (members of the subgenus Sarbecovirus).Limit of detection not yet determined.A single 1 mismatch at probe-binding site identified with the BetaCoV/USA/CA1/2020|EPI_ISL_406034 variant of SARS-CoV-2 (as of 29JAN2020).Probe is in the 3'-5' (reverse complement) direction.Notes: Assay is optimised (as of 24Jan2020).This test has identified a clinical positive case 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 (Wuhan-N; 2019-nCoV-related test) -NOT RECOMMENDED. protocols.io dx.doi.org/10.17504/protocols.io.bchwit7e Copy
Authors: Hyeon Jin Kim, Seong T. Hong
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: We developed a supersensitive capillary reaction kit intended for the direct and qualitative detection of SARS-CoV-2 virus in saliva and nasal swabs. It enables sensitive and specific detection of the SARS-CoV-2 spike antigens in respiratory specimens more accurately than most PCR method within 5 minutes. Our rapid capillary kit will help not only fight but also end COVID-19 by providing the first sensitive self-diagnosis at home as well as POC clinical diagnosis at the health care facilities.
Proper citation: Hyeon Jin Kim, Seong T. Hong 2020. SUPER rapid kit. protocols.io dx.doi.org/10.17504/protocols.io.bkqvkvw6 Copy
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, James Bonfield, Keith James, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, David K. Jackson, Alex Alderton, Ian Johnston
Group: Coronavirus Method Development Community, COG-UK
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) in multiplex PCR. Illumina-compatible sequencing libraries are then made directly from these amplicons in a second PCR step, obviating the need for conventional library preparation. The products of these PCRs are then equivolume pooled and quantitated, prior to sequencing on the Illumina NovaSeq.It is an adaptation of the COVID-19 ARTIC v3 amplicon protocol which can be found here:https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bibtkannBoth the above protocols were adapted from the nCov-2019 sequencing protocol: 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, James Bonfield, Keith James, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, David K. Jackson, Alex Alderton, Ian Johnston 2020. COVID-19 ARTIC v3 Illumina library construction and sequencing protocol - tailed method. protocols.io dx.doi.org/10.17504/protocols.io.bky5kxy6 Copy
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, Carol Scott, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, Alex Alderton, Ian Johnston
Group: Coronavirus Method Development Community, COG-UK
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 offers the benefit of higher density sample processing in 384 format, whilst matching the data quality achieved in 96 format described in the original protocol:https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgxjjxknBoth the above protocols were adapted from the nCov-2019 sequencing protocol: 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, Carol Scott, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, Alex Alderton, Ian Johnston 2020. COVID-19 ARTIC v3 Illumina library construction and sequencing protocol - high throughput 384 format. protocols.io dx.doi.org/10.17504/protocols.io.biefkbbn Copy
Authors: Rushna Quddus, Kaitlynn Snyder, Lauren Altman, Laura Philips, David G. Grier, Andrew D. Hollingsworth, Kent Kirshenbaum
Group: Coronavirus Method Development Community, Grier Group, HolographicDiagnostics, XPRIZE Rapid Covid Testing
Summary: This protocol describes the steps required to perform a holographic immunoassay for SARS-CoV-2 whole virus particles. The assay uses an xSight holographic particle characterization instrument (Spheryx, Inc.) to monitor the diameter of specifically functionalized probe beads. The diameter of the beads increases by a few nanometers as targets bind to the surface. xSight detects and reports this change, yielding an estimate for the concentration of analytes in the sample. The method for holographic immunoassays (applied to antibody binding assays) is described inY. Zagzag, M. F. Soddu, A. D. Hollingsworth and D. G. Grier, Holographic molecular binding assays, Scientific Reports 10, 1932 (2020) and K. Snyder, R. Quddus, A. D. Hollingsworth and K. Kirshenbaum, Holographic Immunoassays: Direct Detection of Antibodies Binding to Colloidal Spheres, submitted for publication (2020).
Proper citation: Rushna Quddus, Kaitlynn Snyder, Lauren Altman, Laura Philips, David G. Grier, Andrew D. Hollingsworth, Kent Kirshenbaum 2020. HolographicDiagnostics: Automated Virus Binding Assay. protocols.io dx.doi.org/10.17504/protocols.io.bkpgkvjw Copy
Authors: Tammy Krylova, Tim Budd, Jerome Nicod, Rupert Beale, Simon Caidan
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 SARS-Cov-2. 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. The first step of the process is sample receipt at the Crick. This SOP describes the inactivation of the virus.
Proper citation: Tammy Krylova, Tim Budd, Jerome Nicod, Rupert Beale, Simon Caidan 2020. Viral inactivation of clinical samples. protocols.io dx.doi.org/10.17504/protocols.io.bfadjia6 Copy
Authors: Isabel Ott, Chantal Vogels, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community
Summary: This protocol details recommendations for collecting and processing saliva for SARS-CoV-2 detection, as used in Wyllie et al. 2020, which details results of testing saliva collected from COVID-19+ inpatients and asymptomatic healthcare workers.COVID-19 inpatients. Saliva samples were self-collected by the patient. Upon waking, patients were asked to avoid food, water and brushing of teeth until the sample was collected. Patients were asked to repeatedly spit into a sterile 90 mL specimen collection cup until roughly a quarter full of liquid (excluding bubbles), before securely closing it. For patients unable to provide saliva (such as those on mechanical ventilation), clinical teams were advised that suction could be used to collect saliva into the cup (or a sputum trap container for improved containment and safety). Collected volumes ranged from 0.5 - 20 mL. All saliva samples were stored at room temperature and transported to the research lab at the Yale School of Public Health within 5 hours of collection, with RNA for SARS-CoV-2 detection extracted within 12 hours of collection. When possible, saliva samples were stored at +4°C, otherwise they were kept at room temperature.Asymptomatic healthcare workers. Asymptomatic healthcare workers were asked to collect ~10 mL of saliva into a sterile specimen collection cup. No specific instructions were given regarding food intake etc prior to collection. Samples were delivered to the research lab at the Yale School of Public Health within 6 hours of collection and stored for up to 6 hours at +4°C until aliquoting for RNA extraction. Samples collected on overnight shifts were stored at +4°C before delivery to the research lab.
Proper citation: Isabel Ott, Chantal Vogels, Nathan Grubaugh, Anne Wyllie 2020. Saliva Collection and RNA Extraction for SARS-CoV-2 Detection. protocols.io dx.doi.org/10.17504/protocols.io.bh6mj9c6 Copy
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 dx.doi.org/10.17504/protocols.io.bi6jkhcn Copy
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 dx.doi.org/10.17504/protocols.io.bd3ji8kn Copy
Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 13 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of Vaccine Candidate against COVID-19 in adults > 18 years of age"This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.Please note that this is an evolving document, to be versioned and updated, based on community feedback and new data.
Proper citation: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores 2020. Publication and Data Sharing Policy (Part 13 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io dx.doi.org/10.17504/protocols.io.bj6akrae Copy
Authors: Josh Quick
Group: ARTIC, Coronavirus Method Development Community, COG-UK
Summary: ARTIC amplicon sequencing protocol for MinION for nCoV-2019This one-pot native barcoding protocol was developed in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC.
Proper citation: Josh Quick 2020. nCoV-2019 sequencing protocol v2 (GunIt). protocols.io dx.doi.org/10.17504/protocols.io.bdp7i5rn Copy
Authors: Moshe Sade-Feldman, Miguel Reyes, Alexandra-Chloe Villani
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: This protocol outlines the steps and methods for blood processing for PBMC to assist the MGH COVID-19 effort.
Proper citation: Moshe Sade-Feldman, Miguel Reyes, Alexandra-Chloe Villani 2020. MGH COVID-19 Effort Blood Processing Protocol for PBMC, Neutrophils and Plasma Isolation. protocols.io dx.doi.org/10.17504/protocols.io.bjhnkj5e Copy
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.
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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 dx.doi.org/10.17504/protocols.io.brrhm536 Copy
Authors: Julia Rossmanith
Group: Coronavirus Method Development Community
Summary: This protocol is for building face shields with closed tops using the print template created by Hanoch Hemmerich on Thingiverse.
Proper citation: Julia Rossmanith 2020. 3D Printed Face Shield with Closed Top. protocols.io dx.doi.org/10.17504/protocols.io.bek6jcze Copy
Authors: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community
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.
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.bii4kcgw Copy
Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 11 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. Ethical Considerations and Informed Consent (Part 11 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io dx.doi.org/10.17504/protocols.io.bj58kq9w Copy
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, Carol Scott, Nicholas Salmon, 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 offers the benefit of higher density sample processing in 384 format, whilst matching the data quality achieved in 96 format described in the original protocol:https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgxjjxknBoth the above protocols were adapted from the nCov-2019 sequencing protocol: 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, Carol Scott, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, Alex Alderton, Ian Johnston 2020. COVID-19 ARTIC v3 Illumina library construction and sequencing protocol - high throughput 384 format. protocols.io dx.doi.org/10.17504/protocols.io.bnidmca6 Copy
Authors: Jessica E. Manning, Jennifer Bohl, Sreyngim Lay, Sophana Chea, Vida Ahyong, Erik Karlsson
Group: Chan Zuckerberg Biohub, Coronavirus Method Development Community
Summary: This protocol was used to enrich for SARS-CoV2 sequencing reads from a confirmed COVID-19 swab sample and attain the full genome using an iSeq100. By using a spiked primer approach with 73 primers spanning the entire SARS-CoV2 genome, we were able to get an average of 15x genome coverage on an iSeq100 with 1.8 million paired end-reads. Here we overview all the steps, from sample extraction, library preparation with a spiked primer enrichment step, and sequencing on an iSeq100. The collaborative effort involved the Manning Lab, NIAID in Phnom Penh, Cambodia, Institut Pasteur Cambodge, Cambodia Ministry of Health, the Chan Zuckerberg Biohub, and the Chan Zuckerberg Initiative.This approach was developed based on the work from Deng et al, Nature Microbiology, January 13, 2020. https://www.nature.com/articles/s41564-019-0637-9
Proper citation: Jessica E. Manning, Jennifer Bohl, Sreyngim Lay, Sophana Chea, Vida Ahyong, Erik Karlsson 2020. SARS-CoV-2 Enrichment Sequencing by Spiked Primer MSSPE method. protocols.io dx.doi.org/10.17504/protocols.io.bc36iyre Copy
Authors: Brett Rasile, Kendra Maas
Group: Coronavirus Method Development Community, 2019-nCoV Wastewater Epidemiology, UConn Mars
Summary: Process
Proper citation: Brett Rasile, Kendra Maas 2021. SARS-CoV-2 Wastewater RNA Concentration and Extraction (Nanotrap® and NucleoMag® RNA Water). protocols.io dx.doi.org/10.17504/protocols.io.bn58mg9w Copy
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