Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage defining mutations (preprint)

The ARTIC protocol uses a multiplexed PCR approach with two primer pools tiling the entire SARS-CoV-2 genome. Primer pool updates are necessary for accurate amplicon sequencing of evolving SARS-CoV-2 variants with novel mutations. The suitability of the ARTIC V4 and updated V4.1 primer scheme was assessed using whole genome sequencing of Omicron from clinical samples using Oxford Nanopore Technology. Analysis of Omicron BA.1 genomes revealed that 93.22% of clinical samples generated improved genome coverage at 50x read depth with V4.1 primers when compared to V4 primers. Additionally, the V4.1 primers improved coverage of BA.1 across amplicons 76 and 88, which resulted in the detection of the variant defining mutations G22898A, A26530G and C26577G. The Omicron BA.2 sub-variant (VUI-22JAN-01) replaced BA.1 as the dominant variant by March 2022, and analysis of 168 clinical samples showed reduced coverage across amplicons 15 and 75. Upon further interrogation of primer binding sites, a mutation at C4321T (present in 163/168, 97% of 30 samples) was identified as a possible cause of complete dropout of amplicon 15. Furthermore, two mutations were identified within the primer binding regions for amplicon 75: A22786C (present in 90% of samples) and C22792T (present in 12.5% of samples). Together, these mutations may result in reduced coverage of amplicon 75 and further primer updates would allow the identification of the two BA.2 defining mutations present in amplicon 75; A22688G and T22679C. This work highlights the need for ongoing surveillance of primer matches as circulating variants evolve and change.

Multi-site validation of an amplicon-based sequencing approach for human monkeypox virus (preprint)

The 2022 multi-country monkeypox outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early human monkeypox virus infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the current outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there is an urgent need for a more sensitive and broadly applicable sequencing approach. Amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for monkeypox virus with amplicon-based and metagenomic sequencing approaches. Upon comparison, we found notably higher genome coverage across the virus genome, particularly in higher PCR cycle threshold (lower DNA titer) samples, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach. By sending out primer pool aliquots to laboratories across the United States and internationally, we validated the primer scheme in 12 public health laboratories with their established Illumina or Oxford Nanopore Technologies sequencing workflows and with different sample types across a range of Ct values. Our findings suggest that amplicon-based sequencing increases the success rate across a wider range of viral DNA concentrations, with the PCR Ct value threshold at which laboratories were able to achieve 80% genome coverage at 10X ranging between Ct 25-33. Therefore, it increases the number of samples where near-complete genomes can be generated and it provides a cost-effective and widely applicable alternative to metagenomics for continued human monkeypox virus genomic surveillance. Importantly, we show that the human monkeypox virus primer scheme can be used with currently implemented amplicon-based SARS-CoV-2 sequencing workflows, with minimal change to the protocol.

Genomics-informed outbreak investigations of SARS-CoV-2 using civet (preprint)

The scale of data produced during the SARS-CoV-2 pandemic has been unprecedented, with more than 5 million sequences shared publicly at the time of writing. This wealth of sequence data provides important context for interpreting local outbreaks. However, placing sequences of interest into national and international context is difficult given the size of the global dataset. Often outbreak investigations and genomic surveillance efforts require running similar analyses again and again on the latest dataset and producing reports. We developed civet (cluster investigation and virus epidemiology tool) to aid these routine analyses and facilitate virus outbreak investigation and surveillance. Civet can place sequences of interest in the local context of background diversity, resolving the query into different 'catchments' and presenting the phylogenetic results alongside metadata in an interactive, distributable report. Civet can be used on a fine scale for clinical outbreak investigation, for local surveillance and cluster discovery, and to routinely summarise the virus diversity circulating on a national level. Civet reports have helped researchers and public health bodies feedback genomic information in the appropriate context within a timeframe that is useful for public health.

Epidemic waves of COVID-19 in Scotland: a genomic perspective on the impact of the introduction and relaxation of lockdown on SARS-CoV-2 (preprint)

The second SARS virus, SARS-CoV-2, emerged in December 2019, and within a month was globally distributed. It was first introduced into Scotland in February 2020 associated with returning travellers and visitors. By March it was circulating in communities across the UK, and to control COVID-19 cases, and prevent overwhelming of the National Health Service (NHS), a 'lockdown' was introduced on 23rd March 2020 with a restriction of people's movements. To augment the public health efforts a large-scale genome epidemiology effort (as part of the COVID-19 Genomics UK (COG-UK) consortium) resulted in the sequencing of over 5000 SARS-CoV-2 genomes by 18th August 2020 from Scottish cases, about a quarter of the estimated number of cases at that time. Here we quantify the geographical origins of the first wave introductions into Scotland from abroad and other UK regions, the spread of these SARS-CoV-2 lineages to different regions within Scotland (defined at the level of NHS Health Board) and the effect of lockdown on virus 'success'. We estimate that approximately 300 introductions seeded lineages in Scotland, with around 25% of these lineages composed of more than five viruses, but by June circulating lineages were reduced to low levels, in line with low numbers of recorded positive cases. Lockdown was, thus, associated with a dramatic reduction in infection numbers and the extinguishing of most virus lineages. Unfortunately since the summer cases have been rising in Scotland in a second wave, with >1000 people testing positive on a daily basis, and hospitalisation of COVID-19 cases on the rise again. Examining the available Scottish genome data from the second wave, and comparing it to the first wave, we find that while some UK lineages have persisted through the summer, the majority of lineages responsible for the second wave are new introductions from outside of Scotland and many from outside of the UK. This indicates that, while lockdown in Scotland is directly linked with the first wave case numbers being brought under control, travel-associated imports (mostly from Europe or other parts of the UK) following the easing of lockdown are responsible for seeding the current epidemic population. This demonstrates that the impact of stringent public health measures can be compromised if following this, movements from regions of high to low prevalence are not minimised.

Genomic epidemiology of SARS-CoV-2 spread in Scotland highlights the role of European travel in COVID-19 emergence (preprint)

SARS-CoV-2, the causative agent of COVID-19, emerged in Wuhan, China in December 2019 and spread rapidly throughout the world. Understanding the introductions of this new coronavirus in different settings may assist control efforts and the establishment of frameworks to support rapid response in future infectious disease outbreaks. We investigated the first four weeks of emergence of the SARS-CoV-2 virus in Scotland after the first case reported on the 1st March 2020. We obtained full genome sequences from 452 individuals with a laboratory-confirmed diagnosis of COVID-19, representing 20% of all cases until 1st April 2020 (n=2310). This permitted a genomic epidemiology approach to study the introductions and spread of the SARS-2 virus in Scotland. From combined phylogenetic and epidemiological analysis, we estimated at least 113 introductions of SARS-CoV-2 into Scotland during this period. Clusters containing multiple sequences suggestive of onward transmission occurred in 48/86 (56%). 42/86 (51%) clusters had no known international travel history indicating undetected introductions. The majority of viral sequences were most closely related to those circulating in other European countries, including Italy, Austria and Spain. Travel-associated introductions of SARS-CoV-2 into Scotland predated travel restrictions in the UK and other European countries. The first local transmission occurred three days after the first case. A shift from travel-associated to sustained community transmission was apparent after only 11 days. Undetected introductions occurred prior to the first known case of COVID-19. Earlier travel restrictions and quarantine measures might have resulted in fewer introductions into Scotland, thereby reducing the number of cases and the subsequent burden on health services. The high number of introductions and transmission rates were likely to have impacted on national contact tracing efforts. Our results also demonstrate that local real-time genomic epidemiology can be used to monitor transmission clusters and facilitate control efforts to restrict the spread of COVID-19. FundingMRC (MC UU 1201412), UKRI/Wellcome (COG-UK), Wellcome Trust Collaborator Award (206298/Z/17/Z - ARTIC Network; TCW Wellcome Trust Award 204802/Z/16/Z Research in contextO_ST_ABSEvidence before this studyC_ST_ABSCoronavirus disease-2019 (COVID-19) was first diagnosed in Scotland on the 1st of March 2020 following the emergence of the causative severe acute respiratory system coronavirus 2 (SARS-CoV-2) virus in China in December 2019. During the first month of the outbreak in Scotland, 2310 positive cases of COVID-19 were detected, associated with 1832 hospital admissions, 207 intensive care admissions and 126 deaths. The number of introductions into Scotland and the source of those introductions was not known prior to this study. Added value of this studyUsing a combined phylogenetic and epidemiological approach following real-time next generation sequencing of 452 SARS-CoV-2 samples, it was estimated that the virus was introduced to Scotland on at least 113 occasions, mostly from other European countries, including Italy, Austria and Spain. Localised outbreaks occurred in the community across multiple Scottish health boards, within healthcare facilities and an international conference and community transmission was established rapidly, before local and international lockdown measures were introduced.