Human tumors are diverse in their natural history and response to treatment, which in part results from genetic and transcriptomic heterogeneity. In clinical practice, single-site needle biopsies are used to sample this diversity, but cancer biomarkers may be confounded by spatiogenomic heterogeneity within individual tumors. Here we investigate clonally expressed genes as a solution to the sampling bias problem by analyzing multiregion whole-exome and RNA sequencing data for 450 tumor regions from 184 patients with lung adenocarcinoma in the TRACERx study. We prospectively validate the survival association of a clonal expression biomarker, Outcome Risk Associated Clonal Lung Expression (ORACLE), in combination with clinicopathological risk factors, and in stage I disease. We expand our mechanistic understanding, discovering that clonal transcriptional signals are detectable before tissue invasion, act as a molecular fingerprint for lethal metastatic clones and predict chemotherapy sensitivity. Lastly, we find that ORACLE summarizes the prognostic information encoded by genetic evolutionary measures, including chromosomal instability, as a concise 23-transcript assay.

Coronaviruses express their structural and accessory genes via a set of subgenomic RNAs, whose synthesis is directed by transcription regulatory sequences (TRSs) in the 5' genomic leader and upstream of each body open reading frame. In SARS-CoV-2, the TRS has the consensus AAACGAAC; upon searching for emergence of this motif in the global SARS-CoV-2 sequences, we find that it evolves frequently, especially in the 3' end of the genome. We show well-supported examples upstream of the Spike gene-within the nsp16 coding region of ORF1b-which is expressed during human infection, and upstream of the canonical Envelope gene TRS, both of which have evolved convergently in multiple lineages. The most frequent neo-TRS is within the coding region of the Nucleocapsid gene, and is present in virtually all viruses from the B.1.1 lineage, including the variants of concern Alpha, Gamma, Omicron and descendants thereof. Here, we demonstrate that this TRS leads to the expression of a novel subgenomic mRNA encoding a truncated C-terminal portion of Nucleocapsid, which is an antagonist of type I interferon production and contributes to viral fitness during infection. We observe distinct phenotypes when the Nucleocapsid coding sequence is mutated compared to when the TRS alone is ablated. Our findings demonstrate that SARS-CoV-2 is undergoing evolutionary changes at the functional RNA level in addition to the amino acid level.

The emergence of successive Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) during 2020 to 2022, each exhibiting increased epidemic growth relative to earlier circulating variants, has created a need to understand the drivers of such growth. However, both pathogen biology and changing host characteristics-such as varying levels of immunity-can combine to influence replication and transmission of SARS-CoV-2 within and between hosts. Disentangling the role of variant and host in individual-level viral shedding of VOCs is essential to inform Coronavirus Disease 2019 (COVID-19) planning and response and interpret past epidemic trends. Using data from a prospective observational cohort study of healthy adult volunteers undergoing weekly occupational health PCR screening, we developed a Bayesian hierarchical model to reconstruct individual-level viral kinetics and estimate how different factors shaped viral dynamics, measured by PCR cycle threshold (Ct) values over time. Jointly accounting for both interindividual variation in Ct values and complex host characteristics-such as vaccination status, exposure history, and age-we found that age and number of prior exposures had a strong influence on peak viral replication. Older individuals and those who had at least 5 prior antigen exposures to vaccination and/or infection typically had much lower levels of shedding. Moreover, we found evidence of a correlation between the speed of early shedding and duration of incubation period when comparing different VOCs and age groups. Our findings illustrate the value of linking information on participant characteristics, symptom profile and infecting variant with prospective PCR sampling, and the importance of accounting for increasingly complex population exposure landscapes when analysing the viral kinetics of VOCs. Trial Registration: The Legacy study is a prospective observational cohort study of healthy adult volunteers undergoing weekly occupational health PCR screening for SARS-CoV-2 at University College London Hospitals or at the Francis Crick Institute (NCT04750356) (22,23). The Legacy study was approved by London Camden and Kings Cross Health Research Authority Research and Ethics committee (IRAS number 286469). The Legacy study was approved by London Camden and Kings Cross Health Research Authority Research and Ethics committee (IRAS number 286469) and is sponsored by University College London Hospitals. Written consent was given by all participants.

Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts’ increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR.

BACKGROUND:SARS-CoV-2 variant Omicron rapidly evolved over 2022, causing three waves of infection due to sub-variants BA.1, BA.2 and BA.4/5. We sought to characterise symptoms and viral loads over the course of COVID-19 infection with these sub-variants in otherwise-healthy, vaccinated, non-hospitalised adults, and compared data to infections with the preceding Delta variant of concern (VOC). METHODS:In a prospective, observational cohort study, healthy vaccinated UK adults who reported a positive polymerase chain reaction (PCR) or lateral flow test, self-swabbed on alternate weekdays until day 10. We compared participant-reported symptoms and viral load trajectories between infections caused by VOCs Delta and Omicron (sub-variants BA.1, BA.2 or BA.4/5), and tested for relationships between vaccine dose, symptoms and PCR cycle threshold (Ct) as a proxy for viral load using Chi-squared (χ2) and Wilcoxon tests. RESULTS:563 infection episodes were reported among 491 participants. Across infection episodes, there was little variation in symptom burden (4 [IQR 3-5] symptoms) and duration (8 [IQR 6-11] days). Whilst symptom profiles differed among infections caused by Delta compared to Omicron sub-variants, symptom profiles were similar between Omicron sub-variants. Anosmia was reported more frequently in Delta infections after 2 doses compared with Omicron sub-variant infections after 3 doses, for example: 42% (25/60) of participants with Delta infection compared to 9% (6/67) with Omicron BA.4/5 (χ2 P < 0.001; OR 7.3 [95% CI 2.7-19.4]). Fever was less common with Delta (20/60 participants; 33%) than Omicron BA.4/5 (39/67; 58%; χ2 P = 0.008; OR 0.4 [CI 0.2-0.7]). Amongst infections with an Omicron sub-variants, symptoms of coryza, fatigue, cough and myalgia predominated. Viral load trajectories and peaks did not differ between Delta, and Omicron, irrespective of symptom severity (including asymptomatic participants), VOC or vaccination status. PCR Ct values were negatively associated with time since vaccination in participants infected with BA.1 (β = -0.05 (CI -0.10-0.01); P = 0.031); however, this trend was not observed in BA.2 or BA.4/5 infections. CONCLUSION:Our study emphasises both the changing symptom profile of COVID-19 infections in the Omicron era, and ongoing transmission risk of Omicron sub-variants in vaccinated adults. TRIAL REGISTRATION:NCT04750356.

The emergence of successive SARS-CoV-2 variants of concern (VOC) during 2020-22, each exhibiting increased epidemic growth relative to earlier circulating variants, has created a need to understand the drivers of such growth. However, both pathogen biology and changing host characteristics - such as varying levels of immunity - can combine to influence replication and transmission of SARS-CoV-2 within and between hosts. Disentangling the role of variant and host in individual-level viral shedding of VOCs is essential to inform COVID-19 planning and response, and interpret past epidemic trends. Using data from a prospective observational cohort study of healthy adult volunteers undergoing weekly occupational health PCR screening, we developed a Bayesian hierarchical model to reconstruct individual-level viral kinetics and estimate how different factors shaped viral dynamics, measured by PCR cycle threshold (Ct) values over time. Jointly accounting for both inter-individual variation in Ct values and complex host characteristics - such as vaccination status, exposure history and age - we found that age and number of prior exposures had a strong influence on peak viral replication. Older individuals and those who had at least five prior antigen exposures to vaccination and/or infection typically had much lower levels of shedding. Moreover, we found evidence of a correlation between the speed of early shedding and duration of incubation period when comparing different VOCs and age groups. Our findings illustrate the value of linking information on participant characteristics, symptom profile and infecting variant with prospective PCR sampling, and the importance of accounting for increasingly complex population exposure landscapes when analysing the viral kinetics of VOCs.

Lung cancer is the leading cause of cancer-associated mortality worldwide 1 . Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS . We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET , ROS1 , ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource.

Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts’ increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR.

ABSTRACT Heterogeneity in SARS-CoV-2 vaccine responses is not understood. Here, we identify four patterns of live-virus neutralizing antibody responses: individuals with hybrid immunity (with confirmed prior infection); rare individuals with low responses (paucity of S1-binding antibodies); and surprisingly, two further groups with distinct serological repertoires. One group – broad responders – neutralize a range of SARS-CoV-2 variants, whereas the other – narrow responders – neutralize fewer, less divergent variants. This heterogeneity does not correlate with Ancestral S1-binding antibody, rather the quality of the serological response. Furthermore, IgD low CD27 - CD137 + B cells and CCR6 + CD4 + T cells are enriched in broad responders before dose 3. Notably, broad responders have significantly longer infection-free time after their third dose. Understanding the control and persistence of these serological profiles could allow personalized approaches to enhance serological breadth after vaccination.

An important component of the UK's early response to the COVID-19 pandemic was increasing SARS-CoV-2 testing capacity across the National Health Service (NHS). At the time, we and others advocated for the repurposing of academic centres to deliver laboratory capacity for testing and screening of asymptomatic health-care workers, to prevent the transmission of SARS-CoV-2.1Black JRM Bailey C Przewrocka J Dijkstra KK Swanton C COVID-19: the case for health-care worker screening to prevent hospital transmission.Lancet. 2020; 395: 1418-1420Summary Full Text Full Text PDF PubMed Scopus (300) Google Scholar In response to a pressing need for testing across London in March, 2020, the Francis Crick Institute rapidly repurposed its laboratory facilities in partnership with University College London Hospitals (UCLH) and the Health Services Laboratory, to create the Crick COVID Testing Pipeline (CCTP). The CCTP provided 680 602 occupational RT-PCR tests from April, 2020, to April, 2022.2Aitken J Ambrose K Barrell S et al.Scalable and robust SARS-CoV-2 testing in an academic center.Nat Biotechnol. 2020; 38: 927-931Crossref PubMed Scopus (22) Google Scholar Same-day results were provided: median turnaround time was 533 min (IQR 456–654; 8·9 h [7·6–10·9]) from receipt to result reporting. All SARS-CoV-2 positive samples were reported to the NHS Test and Trace service, viral genomes were sequenced in-house and submitted to the COVID-19 Genome UK Consortium, and protocols were rapidly shared.2Aitken J Ambrose K Barrell S et al.Scalable and robust SARS-CoV-2 testing in an academic center.Nat Biotechnol. 2020; 38: 927-931Crossref PubMed Scopus (22) Google Scholar The CCTP provided both asymptomatic screening and diagnostic testing for staff in eight NHS trusts and 98 local care homes and rehabilitation facilities up until May, 2021, when NHS testing capacity was realised, covering boroughs with high rates of COVID-19 including Brent, Harrow, Barnet, and Ealing (figure A). Testing was expanded to the Francis Crick Institute in June, 2020, to support essential clinical and research work and continued until May, 2022, under an institutional COVID-19 secure policy.2Aitken J Ambrose K Barrell S et al.Scalable and robust SARS-CoV-2 testing in an academic center.Nat Biotechnol. 2020; 38: 927-931Crossref PubMed Scopus (22) Google Scholar Here, we performed an analysis of testing data with a view to understanding both the effect of hospital trust screening policies and the Francis Crick Institute's in-person work policy to inform future pandemic planning. Overall, 7316 positive infections were detected from 680 602 tests (1%) between April 1, 2020, and April 14, 2022 (figure B). Across the CCTP, the rolling 7-day incidence (7-day mean number of positive tests per 1000 tests taken per site) showed that infections in health-care workers mirrored the incidence of national trends (figure C).3Vöhringer HS Sanderson T Sinnott M et al.Genomic reconstruction of the SARS-CoV-2 epidemic in England.Nature. 2021; 600: 506-511Crossref PubMed Scopus (44) Google Scholar We captured variants of concern (figure D–E)—often ahead of their designation by WHO (figure F)—whereby alpha (B.1.1.7) was first detected 33 days before, delta (B.1.617.2) 27 days before, and omicron (BA.1) 5 days following its designation. We first explored the interactions across the CCTP between positive test rates, viral loads, and different testing policies adopted by NHS trusts between 2020 and 2021 (appendix pp 10–11). We compared the 7-day rolling infection incidence between sites (appendix pp 10–11) and found, as expected, that the incidence was highest when testing was predominantly offered to symptomatic staff, including at sites managed by the London Northwest University Hospitals NHS Trust and The Royal Free Hospital NHS Trust. However, despite a low 7-day rolling infection incidence, 40·8% of all positive tests were from UCLH and Royal Marsden Hospitals; both sites offered asymptomatic testing to support the delivery of COVID-19 secure cancer and surgical services. These sites detected a substantial number of additional infections. To investigate the differences between sites operating symptomatic and asymptomatic testing policies, we calculated an incidence for each site by adjusting for the organisation size (defined by the number of employees stated in their 2020–21 annual reports), and then calculated an incidence rate ratio (IRR) for each site over the first 4 months of testing, defined as the daily incidence of positive tests for each site, divided by the daily incidence estimates for London's population.4Greater London AuthorityCoronavirus (COVID-19) weekly update.https://data.london.gov.uk/dataset/coronavirus--covid-19--casesDate accessed: November 1, 2022Google Scholar We compared the IRR between primarily symptomatic and asymptomatic testing sites, including those following NHS guidelines to create COVID-19 secure clinical areas.5UK Health Security AgencyInfection prevention and control for seasonal respiratory infections in health and care settings (including SARS-CoV-2) for winter 2021 to 2022.https://www.gov.uk/government/publications/wuhan-novel-coronavirus-infection-prevention-and-control/covid-19-guidance-for-maintaining-services-within-health-and-care-settings-infection-prevention-and-control-recommendationsDate accessed: November 3, 2021Google Scholar During the first 4 months of testing, the incidence of cases at NHS sites (UCLH and Royal Marsden Hospitals) that offered primarily asymptomatic SARS-CoV-2 testing was 7·5 and 13 times respectively that of the London population (appendix pp 10–11) and NHS sites continued to report positive tests consistently above 5 times the background population symptomatic rate until the second week of July, 2020 (appendix pp 3–4). Asymptomatic screening is designed to detect early infections, reducing transmission with self isolation.1Black JRM Bailey C Przewrocka J Dijkstra KK Swanton C COVID-19: the case for health-care worker screening to prevent hospital transmission.Lancet. 2020; 395: 1418-1420Summary Full Text Full Text PDF PubMed Scopus (300) Google Scholar We compared the distribution of PCR cycle threshold (Ct) value between the two types of sites as a well recognised proxy measure of viral load and thus, infectiousness.6Killingley B Mann AJ Kalinova M et al.Safety, tolerability and viral kinetics during SARS-CoV-2 human challenge in young adults.Nat Med. 2022; 28: 1031-1041Crossref PubMed Scopus (190) Google Scholar We found the median Ct was lower (corresponding with a higher viral load) in symptomatic test sites (Ct 21·2 [IQR 8·06], n=1458), compared with asymptomatic sites (Ct 25·7 [10·1], n=3322, p<0·0001; appendix pp 3–4). Ct values typically peak just before or around symptom onset and symptomatic-only testing might be biased towards the post-peak period; therefore, asymptomatic testing might further detect a subset of people with infectious virus at or before the Ct peak.6Killingley B Mann AJ Kalinova M et al.Safety, tolerability and viral kinetics during SARS-CoV-2 human challenge in young adults.Nat Med. 2022; 28: 1031-1041Crossref PubMed Scopus (190) Google Scholar To capture peak Ct values and control for different Ct ranges across sites with different testing policies, we compared the cumulative occurrence of positive tests between asymptomatic and symptomatic sites in a subset of swabs with a Ct value of less than 15 (appendix pp 10–11). Within this subset, we found that asymptomatic sites identified infections at lower Ct values, suggesting enhanced detection of these highly infectious individuals. We then analysed the Francis Crick Institute, whereby to support a rapid return to in-person working, we implemented a COVID-19 secure workplace policy.7de Quetteville H How Sir Paul Nurse got 1200 scientists safely back to work with the 'easy' test and trace method.https://www.telegraph.co.uk/health-fitness/mind/sir-paul-nurse-got-1200-scientists-safely-back-work-easy-test/Date: Aug 10, 2020Date accessed: November 3, 2021Google Scholar With an asymptomatic testing policy, entry to the institute was contingent on negative testing within the previous 8 days, internal contact tracing following positive tests, distancing, and face coverings were enforced by social compliance. The institute remained open, while the weekly test positivity remained less than 1%. Analysis of building occupancy data showed mean 7-day occupancy was maintained at over 60% of peak attendance for the overwhelming majority of days (356 [84%] of 423 days; appendix pp 10–11), reaching 75% peak attendance by September, 2020. The 1% threshold was met on Oct 22, 2020, following the spread of the EU1 variant (B.1.177), and again on Dec 8, 2021, as a result of the omicron variant (BA.1, appendix pp 5–6). The Francis Crick Institute implemented a stricter testing protocol resulting in flatter peaks of infection in employees than were otherwise observed within our local borough of Camden (appendix pp 10–11). Taken together, we show that it was possible to repurpose laboratory facilities and integrate existing clinical and laboratory expertise to set up a comprehensive testing facility in an academic institution at pace,2Aitken J Ambrose K Barrell S et al.Scalable and robust SARS-CoV-2 testing in an academic center.Nat Biotechnol. 2020; 38: 927-931Crossref PubMed Scopus (22) Google Scholar serving as a lifeboat laboratory and delivering testing for the NHS in advance of the planned national programme. The CCTP was designed to protect both staff and patients with testing; we show important heterogeneity in the uptake of testing by hospitals. Health-care workers are susceptible to the occupational acquisition of SARS-CoV-2 despite infection control practices.8Houlihan CF Vora N Byrne T et al.Pandemic peak SARS-CoV-2 infection and seroconversion rates in London frontline health-care workers.Lancet. 2020; 396: e6-e7Summary Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Meredith LW Hamilton WL Warne B et al.Rapid implementation of SARS-CoV-2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study.Lancet Infect Dis. 2020; 20: 1263-1271Summary Full Text Full Text PDF PubMed Scopus (259) Google Scholar Estimates of true asymptomatic infections vary, but up to 40% of all COVID-19 infections might be asymptomatic.10Ma Q Liu J Liu Q et al.Global percentage of asymptomatic SARS-CoV-2 infections among the tested population and individuals with confirmed COVID-19 diagnosis: a systematic review and meta-analysis.JAMA Netw Open. 2021; 4e2137257Crossref Scopus (251) Google Scholar Although our study is restricted by its observational design, our data suggest that asymptomatic testing strategies captured a considerable number of additional infections, particularly in the early phase of the pandemic, and supports NHS England Infection Prevention and Control advice on the use of asymptomatic testing with non-pharmaceutical interventions to maintain green sites to provide care for clinically susceptible patients.11UK Health Security AgencyNational infection prevention and control manual (NIPCM) for England.https://www.england.nhs.uk/national-infection-prevention-and-control-manual-nipcm-for-england/Date: 2021Date accessed: November 3, 2021Google Scholar Our data also provide a unique insight into workplace exposure risk and screening strategies outside of hospital settings, with most studies focusing on higher risk settings such as homeless shelters, prisons, schools, and cruise ships. UK Government recommendations on non-clinical workplace safety during COVID-19 centred on ventilation, social distancing, encouraging vaccination, and self isolation. The Francis Crick Institute's enforced testing policy supported a safe workplace, potentially minimising infection peaks during the delta wave in summer 2021. In the absence of data from similar organisations, our study suggests that asymptomatic screening with non-pharmaceutical interventions are an important addition to guidelines on workplace safety. Overall, this study provides a blueprint for future NHS–academic partnerships to follow. With active planning underway on national preparedness for the next potential pandemic, our work highlights the importance of prioritising testing—including regular asymptomatic testing of key workers including NHS and care home staff—during the first phase of the pandemic response. Sequencing data for all positive samples are publicly available through COG-UK resources. CB, TS, HT, JGo, RGi, JN, DLVB, and ECW accessed and verified the data. CB, ECW, DLVB, SGan, and CSw were responsible for the decision to submit the Correspondence for publication. CB and TS were responsible for formal analysis, investigation, methodology, visualisation, writing the original draft, and conceptualisation. HT was involved in the investigation, methodology, visualisation, manuscript review and editing, and conceptualisation. JGo was responsible for software, methodology, formal analysis, and data curation. JRMB performed formal analysis and validation. JGan was responsible for the methodology, resources, data curation, and project administration. GY performed formal analysis. RGo was responsible for software, resources, and data curation. ASF, SW, DJJ, LC, VD, OO'N, MC, DS, MF, AE, JP-L, AR, JA, NO'R, SC, MYW, PAW, and CSa were involved with the methodology, resources, data curation, and project administration. EJC was involved with software and project administration. SH, JF, and KA helped with supervision, software, and methodology. MHo performed project administration and supervision. AJ was responsible for methodology, resources, data curation, and project administration. CH, EN, MHu, RM, DH, PP, TC, RGi, JM, NVA, ST, RB, ML, and SB managed resources, data curation, and project administration. BW and SGam handled funding acquisition and project administration with SGam also providing supervision. JN was involved with project administration, supervision, and methodology. SGan performed supervision, funding acquisition, methodology, project administration, writing, review, and editing. DLVB was responsible for supervision, methodology, formal analysis, visualisation, conceptualisation, and writing the original draft. ECW performed supervision, investigation, data curation, conceptualisation, writing, review, and editing. CSw was responsible for supervision, funding acquisition, conceptualisation, project administration, writing, review, and editing. This research was funded in whole, or in part, by the Wellcome Trust (FC011104, FC011233, FC001030, FC001159, FC001827, FC001078, FC001099, and FC001169). TS is supported by a Sir Henry Wellcome Postdoctoral Fellowship from the Wellcome Trust grant 210918/Z/18/Z. Unrelated to this Correspondence, CSw reports grants from BMS, Ono-Pharmaceuticals, Boehringer Ingelheim, Roche-Ventana, Pfizer, and Archer Dx; personal fees from Genentech, Sarah Canon Research Institute, Medicxi, Metabomed, Bicycle Therapeutics, GRAIL, Amgen, AstraZeneca, BMS, Illumina, GlaxoSmithKline, MSD, and Roche-Ventana; and stock options from Apogen Biotech, Epic Biosciences, GRAIL, Achilles Therapeutics, and Bicycle Therapeutics. We thank Sir Paul Nurse, Jules Marczack, Bobbi Clayton, Gita Mistry, and all the research staff who volunteered to work on the COVID-19 testing pipeline at the Francis Crick Institute. We also thank the staff of the National Institute for Health and Care Research Clinical Research Facility at University College London Hospitals NHS Foundation Trust including Dr Mike Brown, Martin Bruce, Kirsty Adams, Miguel Alvarez, Marivic Ricamara, and Dr Mike Gandy at the Health Services Laboratory. Download .pdf (2.92 MB) Help with pdf files Supplementary appendix COVID-19: the case for health-care worker screening to prevent hospital transmissionThe outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has placed unprecedented strain on health-care services worldwide, leading to more than 100 000 deaths worldwide, as of April 15, 2020.1 Full-Text PDF

Background SARS-CoV-2 variant Omicron rapidly evolved over 2022, causing three waves of infection due to sub-variants BA.1, BA.2 and BA.4/5. We sought to characterise symptoms and viral loads over the course of COVID-19 infection with these sub-variants in otherwise-healthy, vaccinated, non-hospitalised adults, and compared data to infections with the preceding Delta variant of concern (VOC).Methods In a prospective, observational cohort study, healthy vaccinated UK adults who reported a positive PCR or lateral flow test, self-swabbed on alternate days until day 10. We compared symptoms and viral load trajectories between infections caused by VOCs Delta and Omicron (sub-variants BA.1, BA.2 and BA.4/5), and tested for relationships between vaccine dose, symptoms and PCR Ct value as a proxy for viral load.Results 555 infection episodes were reported among 483 participants. Across VOCs, symptom burden and duration were similar, however symptom profiles differed among infections caused by Delta compared to Omicron sub-variants; symptoms of all Omicron sub-variants BA.1, BA.2 and BA.4/5 were very similar. Anosmia was reported in 7-13% of participants with Omicron sub-variants, compared to 25/60 (42%) with Delta infection (P= 1.31e-08 or 1.03e-05 or 5.63e-05; χ2 test d2+Delta vs. Omicron BA.1 or vs. BA.2, or BA.5, respectively), fever was more common with Omicron BA.5 (30/55, 55%) than Delta (20/60, 33%) (p 0.03). Amongst infections with all Omicron sub-variants, symptoms of coryza, fatigue, cough and myalgia predominated. Viral load trajectories and peaks did not differ between Delta, and Omicron, irrespective of symptom severity (including asymptomatic participants), VOC or vaccination status. Ct values were negatively associated with time since vaccination in participants infected with BA.1; however, this trend was not observed in BA.2/BA.4/5 infections.Conclusion Our study emphasises both the changing symptom profile of COVID-19 infections in the Omicron era, and ongoing transmission risk of Omicron sub-variants in vaccinated adults.Trial registration NCT04750356### Competing Interest StatementCSw reports interests unrelated to this Correspondence: grants from BMS, Ono-Pharmaceuticals, Boehringer-Ingelheim, Roche-Ventana, Pfizer and Archer Dx, unrelated to this Correspondence; personal fees from Genentech, Sarah Canon Research Institute, Medicxi, Bicycle Therapeutics, GRAIL, Amgen, AstraZeneca, BMS, Illumina, GlaxoSmithKline, MSD, and Roche-Ventana, unrelated to this Correspondence; and stock options from Apogen Biotech, Epic Biosciences, GRAIL, and Achilles Therapeutics, unrelated to this Correspondence. DLVB reports grants from AstraZeneca unrelated to this Correspondence. All other authors declare no competing interests.### Funding StatementThis work was supported by UCLH/UCL who received a proportion of funding from the National Institute for Health Research (NIHR) University College London Hospitals Department of Health NIHR Biomedical Research Centre (BRC). EW VL and BW are supported by the Centre funding scheme. This work was supported jointly by the BRC and core funding from the Francis Crick Institute which receives its funding from Cancer Research UK the UK Medical Research Council and the Wellcome Trust. This research was funded in whole or in part by the Wellcome Trust. For the purpose of Open Access the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. DLVB is additionally supported by the Genotype to Phenotype National Virology Consortium via UK Research and Innovation and the UK Medical Research Council and TWR and AJK are additionally supported by the Wellcome Trust. ### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The Legacy study was approved by London Camden and Kings Cross Health Research Authority Research and Ethics committee (IRAS number 286469)I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present work are contained in the manuscript and will be made openly available online after peer reviewAll data produced in the present work are contained in the manuscript and will be made openly available online after peer review

Two mutations occurred in SARS-CoV-2 early during the COVID-19 pandemic that have come to define circulating virus lineages 1 : first a change in the spike protein (D614G) that defines the B.1 lineage and second, a double substitution in the nucleocapsid protein (R203K, G204R) that defines the B.1.1 lineage, which has subsequently given rise to three Variants of Concern: Alpha, Gamma and Omicron. While the latter mutations appear unremarkable at the protein level, there are dramatic implications at the nucleotide level: the GGG→AAC substitution generates a new Transcription Regulatory Sequence (TRS) motif, driving SARS-CoV-2 to express a novel subgenomic mRNA (sgmRNA) encoding a truncated C-terminal portion of nucleocapsid (N.iORF3), which is an inhibitor of type I interferon production. We find that N.iORF3 also emerged independently within the Iota variant, and further show that additional TRS motifs have convergently evolved to express novel sgmRNAs; notably upstream of Spike within the nsp16 coding region of ORF1b, which is expressed during human infection. Our findings demonstrate that SARS-CoV-2 is undergoing evolutionary changes at the functional RNA level in addition to the amino acid level, reminiscent of eukaryotic evolution. Greater attention to this aspect in the assessment of emerging strains of SARS-CoV-2 is warranted.

In this cohort study, we aim to compare outcomes from coronavirus disease 2019 (COVID-19) in people with severe epilepsy and other co-morbidities living in long-term care facilities which all implemented early preventative measures, but different levels of surveillance. During 25-week observation period (16 March-6 September 2020), we included 404 residents (118 children), and 1643 caregivers. We compare strategies for infection prevention, control, and containment, and related outcomes, across four UK long-term care facilities. Strategies included early on-site enhancement of preventative and infection control measures, early identification and isolation of symptomatic cases, contact tracing, mass surveillance of asymptomatic cases and contacts. We measured infection rate among vulnerable people living in the facilities and their caregivers, with asymptomatic and symptomatic cases, including fatality rate. We report 38 individuals (17 residents) who tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive, with outbreaks amongst residents in two facilities. At Chalfont Centre for Epilepsy (CCE), 10/98 residents tested positive: two symptomatic (one died), eight asymptomatic on weekly enhanced surveillance; 2/275 caregivers tested positive: one symptomatic, one asymptomatic. At St Elizabeth's (STE), 7/146 residents tested positive: four symptomatic (one died), one positive during hospital admission for symptoms unrelated to COVID-19, two asymptomatic on one-off testing of all 146 residents; 106/601 symptomatic caregivers were tested, 13 positive. In addition, during two cycles of systematically testing all asymptomatic carers, four tested positive. At The Meath (TM), 8/80 residents were symptomatic but none tested; 26/250 caregivers were tested, two positive. At Young Epilepsy (YE), 8/80 children were tested, all negative; 22/517 caregivers were tested, one positive. Infection outbreaks in long-term care facilities for vulnerable people with epilepsy can be quickly contained, but only if asymptomatic individuals are identified through enhanced surveillance at resident and caregiver level. We observed a low rate of morbidity and mortality, which confirmed that preventative measures with isolation of suspected and confirmed COVID-19 residents can reduce resident-to-resident and resident-to-caregiver transmission. Children and young adults appear to have lower infection rates. Even in people with epilepsy and multiple co-morbidities, we observed a high percentage of asymptomatic people suggesting that epilepsy-related factors (anti-seizure medications and seizures) do not necessarily lead to poor outcomes. (C) 2020 Elsevier Inc. All rights reserved.

Patients with cancer have higher COVID-19 morbidity and mortality. Here we present the prospective CAPTURE study, integrating longitudinal immune profiling with clinical annotation. Of 357 patients with cancer, 118 were SARS-CoV-2 positive, 94 were symptomatic and 2 died of COVID-19. In this cohort, 83% patients had S1-reactive antibodies and 82% had neutralizing antibodies against wild type SARS-CoV-2, whereas neutralizing antibody titers against the Alpha, Beta and Delta variants were substantially reduced. S1-reactive antibody levels decreased in 13% of patients, whereas neutralizing antibody titers remained stable for up to 329 days. Patients also had detectable SARS-CoV-2-specific T cells and CD4 + responses correlating with S1-reactive antibody levels, although patients with hematological malignancies had impaired immune responses that were disease and treatment specific, but presented compensatory cellular responses, further supported by clinical recovery in all but one patient. Overall, these findings advance the understanding of the nature and duration of the immune response to SARS-CoV-2 in patients with cancer.

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP that is robust, reliable, repeatable, specific, and inexpensive.

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP that is robust, reliable, repeatable, specific, and inexpensive.

The SARS-CoV-2 B.1.617.2 Variant of Concern (VOC), first detected in India, is now dominant in the UK, having rapidly1Public Health EnglandSARS-CoV-2 variants of concern and variants under investigation in England, Technical briefing 13.https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/990177/Variants_of_Concern_VOC_Technical_Briefing_13_England.pdfDate: May 27, 2021Date accessed: June 2, 2021Google Scholar displaced the B.1.1.7 strain2Rambaut A Loman N Pybus O et al.Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations.https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563Date: Feb 4, 2021Date accessed: June 2, 2021Google Scholar that emerged in the UK with the second COVID-19 wave in late 2020. The efficacy of currently licensed COVID-19 vaccines against B.1.617.2 is unknown; although it possesses 12 mutations in its spike protein relative to the wildtype SARS-CoV-2 first detected in Wuhan, China, in December, 2019, B.1.617.2 lacks mutations at amino acid positions 501 or 484 in its ACE2 receptor-binding domain, commonly associated with VOCs (appendix p 2) or escape from neutralising antibodies (NAbs). To determine vaccine-induced NAb escape by B.1.617.2 and compare activity to previous strains with existing estimates for population-based vaccine efficacy, we carried out an initial analysis of the Legacy study, established in January, 2021, by University College London Hospital and the Francis Crick Institute in London, UK, to track serological responses to vaccination in prospectively recruited staff volunteers (appendix p 6). A detailed description of the methods, including the clinical cohort, virus culture conditions, genetic sequencing, and neutralisation assays, and the statistical analysis are available in the appendix (p 8). The Legacy study was approved by London Camden and Kings Cross Health Research Authority Research and Ethics committee (IRAS number 286469) and sponsored by University College London. Using a high-throughput live-virus SARS-CoV-2 neutralisation assay (performance data are shown in the appendix p 3), we determined NAb titres (NAbTs) in 250 participants (median age 42 years [IQR 33–52]) after either one dose (n=149; median time after first dose=30 days [IQR 23–38]) or two doses (n=159; median time after second dose=28 days [IQR 21–37]) of BNT162b2 (Pfizer–BioNTech) against five SARS-CoV-2 strains: a strain with the original spike sequence (Wild-type); a strain with an Asp614Gly mutation isolated during the first wave of infection in the UK, in 2020 (D614G); and VOCs B.1.617.2, B.1.351 (first detected in South Africa in late 2020), and B.1.1.7. Two doses of BNT162b2 elicited ELISA-detected anti-Wild-type spike antibodies in all participants, and NAb activity against all strains, including the three VOCs tested, in all except six (3%) and nine (5%) of 159 participants who lacked NAb activity against B.1.617.2 and B.1.351, respectively (appendix p 2). NAbTs of sera correlated well between Wild-type and variants (appendix p 2; RS>0·82, p<2 × 10−16), as well as between VOCs (B.1.617.2 vs B.1.351: RS=0·85, p<2 × 10−16). However, NAbTs were 5·8-fold reduced against B.1.617.2 relative to Wild-type (95% CI 5·0–6·9), significantly more reduced than against B.1.1.7 (2·6-fold vs Wild-type, 95% CI 2·2–3·1), and on a similar order to the reduction observed against B.1.351 (4·9-fold vs Wild-type, 95% CI 4·2–5·7). Notably, across all variants, increased age significantly correlated with reduced NAbT (appendix p 2; −0·3340), two participants' NAbTs against VOCs B.1.617.2 and B.1.351 dropped below 40 on their later study visit about 3 months after their second BNT162b2 dose. To maximise population coverage, the UK extended the interval between the two BNT162b2 doses. Although this might have had a limited impact of protection against parental SARS-CoV-2 strains or the B.1.1.7 variant, the potential impact on protection from other VOCs is poorly understood. We found that neutralisation of VOCs was markedly different after only one dose of BNT162b2 (appendix p 2): although 177 (95%) of 186 participants tested positive for anti-spike antibodies by ELISA and mounted a detectable NAb response against Wild-type (median IC50=68 [IQR 42–140]) and D614G (median IC50=71 [IQR 46–111]), median NAbTs against all VOCs were below the quantitative limit of detection. Stratification of NAbTs into three groups (IC50 low [<40], medium [40–256], high [>256]) and assessment of the significance of the shift in their distribution relative to Wild-type by ordered logistical regression was more informative (appendix p 2). Whereas only 39 (21%) of 186 samples had low NAbTs against Wild-type, this proportion rose to 50% against B.1.1.7 (p=1·7 × 10−6) and further to 75% against B.1.351 (p<3 × 10−16) and 68% against B.1.617.2 (p<5 × 10−16). Notably, the downwards shift in titres was also significant when compared to B.1.1.7 for B.1.351 (p=3·7 × 10−4) and B.1.617.2 (p=1·2 × 10−5), confirming reduced NAb activity against B.1.617.2 relative to the present B.1.1.7 strain after one vaccine dose. Notably, participants with low NAbTs tend to be older than those who produced medium or high responses (appendix p 2), and logistical regression analysis suggests age is a significant factor in reduced NAbTs, independent of strain in our samples (appendix p 7; p=0.006), following a single dose of BNT162b2. These data, together with epidemiological data of B.1.617.2 growth, raise the possibility that this VOC presents a dual challenge of reduced vaccine efficacy akin to the B.1.351 VOC, and increased transmissibility beyond the B.1.1.7 VOC. The impact of such a change is challenging to predict: it remains difficult to assess precisely to what extent the reduction in NAbTs we observe will impact vaccine efficacy and increase disease severity in a vaccinated population, especially given the multiple factors that contribute to this process, such as long-lived humoral immunity.3Turner JS Kim W Kalaidina E et al.SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans.Nature. 2021; (published online May 24.)https://doi.org/10.1038/s41586-021-03647-4Crossref Scopus (339) Google Scholar Nevertheless, a recent analysis of available NAb and vaccine efficacy data4Khoury DS Cromer D Reynaldi A et al.Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection.Nat Med. 2021; (published online May 17.)https://doi.org/10.1038/s41591-021-01377-8Crossref PubMed Scopus (2438) Google Scholar has attempted to establish correlates of protection against earlier strains of SARS-CoV-2 and, in the context of this model, our data suggest that most participants that received two doses of BNT162b2 would be protected against B.1.617.2 infection and associated disease—consistent with preliminary data5Bernal JL Andrews N Gower C et al.Effectiveness of COVID-19 vaccines against the B.1.617.2 variant.KHub. 2021; (published online May 24.) (preprint).https://doi.org/10.1101/2021.05.22.21257658Google Scholar inferring vaccine efficacy against B.1.617.2 in the UK based on rates of S-gene target failure during quantitative RT-PCR testing. With increasing case numbers and the proportion of sequencing-confirmed B.1.617.2 cases, coupled with wider availability of WHO International Standards and Reference Panels to standardise NAbTs across laboratories, we expect that improved vaccine efficacy estimates will allow more precise modelling of correlates of protection in the coming months. However, it is worth highlighting that in the case of two BNT162b2 doses, our cohort of generally healthy, relatively young, recently vaccinated, and mostly single-ethnicity individuals presents a reasonable best-case scenario for NAb activity against SARS-CoV-2 variants. Indeed, regardless of the absolute vaccine efficacy requirements, peak NAbTs are significantly reduced against VOCs B.1.617.2 and B.1.351 compared with NAbTs against earlier variants, and consequently, vaccine efficacy on an individual or sub-population level will become more sensitive to reductions in NAbTs occurring as a result of factors aside from virus strain (appendix p 5), providing a basis to understand observed vaccine efficacy failure in other combinations of vaccine and target population.6Madhi SA Baillie V Cutland CL et al.Efficacy of the ChAdOx1 nCoV-19 Covid-19 vaccine against the B.1.351 variant.N Engl J Med. 2021; 384: 1885-1898Crossref PubMed Scopus (864) Google Scholar In the case of single-dose recipients, our data show that NAbTs are significantly lower against B.1.617.2 and B.1.351 VOCs relative to B.1.1.7, implying that although a single dose might still afford considerably more protection than no vaccination, single-dose recipients are likely to be less protected against these SARS-CoV-2 variants. These data therefore suggest that the benefits of delaying the second dose, in terms of wider population coverage and increased individual NAbTs after the second dose,7Parry H Bruton R Stephens C et al.Extended interval BNT162b2 vaccination enhances peak antibody generation in older people.MedRxiv. 2021; (published online May 17.) (preprint).https://doi.org/10.1101/2021.05.15.21257017Google Scholar must now be weighed against decreased efficacy in the short-term, in the context of the spread of B.1.617.2. Worldwide, our data highlight the ongoing need to increase vaccine supply to allow all countries to extend second-dose protection as quickly as possible. In the longer term, we note that both increased age and time since the second dose of BNT162b2 significantly correlate with decreased NAb activity against B.1.617.2 and B.1.351—both of which are also characteristic of the population in the UK at highest risk of severe COVID-19 (ie, older and vaccinated earlier), independent of other existing factors such as compromised immune status or comorbidity, or geographic-specific responses to vaccination. Consequently, further booster immunisations of JCVI Priority Groups in the UK and similar groups in other counties, as well as others with lower vaccine-induced NAbTs than the cohort of BNT162b2 recipients studied here (ideally with modified vaccines that induce NAbs that broadly neutralise emerging VOCs) are more likely to be required to maintain the highest levels of NAbs in regions where B.1.617.2 or other equally NAb-resistant strains become prevalent. CSw reports grants from BMS, Ono-Pharmaceuticals, Boehringer-Ingelheim, Roche-Ventana, Pfizer and Archer Dx, unrelated to this Correspondence; personal fees from Genentech, Sarah Canon Research Institute, Medicxi, Bicycle Therapeutics, GRAIL, Amgen, AstraZeneca, BMS, Illumina, GlaxoSmithKline, MSD, and Roche-Ventana, unrelated to this Correspondence; and stock options from Apogen Biotech, Epic Biosciences, GRAIL, and Achilles Therapeutics, unrelated to this Correspondence. All other authors declare no competing interests. ECW, MW, SG, and DLVB contributed equally. GKa, CSw, SGan, and DLVB are joint senior authors. RB and DLVB are members of the Genotype-to-Phenotype UK National Virology Consortium. Funding details and acknowledgments can be found in the appendix. All data (anonymised) and full R code to produce all figures and statistical analysis presented in this Correspondence are available online on Github. Download .pdf (4.14 MB) Help with pdf files Supplementary appendix Three-dose vaccination elicits neutralising antibodies against omicronOmicron, the SARS-CoV-2 B.1.1.529 variant of concern (VOC), was first detected in southern Africa in November, 2021, and its BA.1 sub-lineage is now dominant in the UK. Omicron BA.1 contains 32 coding changes in its spike protein (appendix p 2), and it is unclear to what extent its spread is driven by an intrinsic increase in transmissibility or escape from previous infection-induced and vaccine-induced immunity. Full-Text PDF

Combining samples for genetic association is standard practice in human genetic analysis of complex traits, but is rarely undertaken in rodent genetics. Here, using 23 phenotypes and genotypes from two independent laboratories, we obtained a sample size of 3076 commercially available outbred mice and identified 70 loci, more than double the number of loci identified in the component studies. Fine-mapping in the combined sample reduced the number of likely causal variants, with a median reduction in set size of 51%, and indicated novel gene associations, including Pnpo, Ttll6, and GM11545 with bone mineral density, and Psmb9 with weight. However, replication at a nominal threshold of 0.05 between the two component studies was low, with less than one-third of loci identified in one study replicated in the second. In addition to overestimates in the effect size in the discovery sample (Winner's Curse), we also found that heterogeneity between studies explained the poor replication, but the contribution of these two factors varied among traits. Leveraging these observations, we integrated information about replication rates, study-specific heterogeneity, and Winner's Curse corrected estimates of power to assign variants to one of four confidence levels. Our approach addresses concerns about reproducibility and demonstrates how to obtain robust results from mapping complex traits in any genome-wide association study.

Purpose of examination / Clinical relevance At 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 extraction of RNA from the inactivated sample. Principles of Examination This procedure involves extraction of COVID-19 RNA from inactivated virus samples that have been aliquoted into plates. This protocol follows a method developed in-house that is automated on the Biomek FX liquid handling platform. All reagents and buffers prepared in house are batch tested and certified.

Purpose of examination / Cl inical re levance At 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 transfer of inactivated virus into plates for RNA extraction. Principles of Examination The assay uses the Hamilton robot to transfer 150 μl of 93 samples of inactivated viral media samples from Barcoded 2 ml tubes (Cat no. 72.694.005) to Barcoded 96 well Nunc plates (Cat no. 260251) maintaining sample identity. -The authors wish to thank Heather Ringrose for support with the Hamilton liquid handlingworkstation. This protocol is part of the Crick COVID-19 RT-PCR Testing Pipeline collection. THIS DOCUMENT MUST BE READ AND UNDERSTOOD BY STAFF USING IT AND DOCUMENTED EVIDENCE PROVIDED THEREOF. EXTERNAL LINK https://doi.org/10.1038/s41587-020-0588-y THIS PROTOCOL ACCOMPANIES THE FOLLOWING PUBLICATION Aitken, J., Ambrose, K., Barrell, S. et al. Scalable and robust SARS-CoV-2 testing in an academic center. Nat 1 05/16/2020 Cita tion : Efthymios Fidanis, Sophia Ward, Laura Cubitt, Amelia Edwards, Debbie Hughes, Michael Hubank, Jerome Nicod (05/16/2020). Automated transfer of inactivated virus samples from individual 2ml tubes to 96-well plate. https://dx.doi.org/10.17504/protocols.io.bfb2jiqe This is an open access protocol distributed under the terms of the Crea tive Com m ons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Biotechnol (2020). https://doi.org/10.1038/s41587-020-0588-y DOI dx.doi.org/10.17504/protocols.io.bfb2jiqe PROTOCOL CITATION Efthymios Fidanis, Sophia Ward, Laura Cubitt, Amelia Edwards, Debbie Hughes, Michael Hubank, Jerome Nicod 2020. Automated transfer of inactivated virus samples from individual 2ml tubes to 96-well plate. protocols.io https://dx.doi.org/10.17504/protocols.io.bfb2jiqe MANUSCRIPT CITATION please remember to cite the following publication along with this protocol Aitken, J., Ambrose, K., Barrell, S. et al. Scalable and robust SARS-CoV-2 testing in an academic center. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0588-y EXTERNAL LINK https://doi.org/10.1038/s41587-020-0588-y COLLECTIONS The Crick COVID-19 RT-PCR Testing Pipel ine