Abstract As part of preparedness activities supporting pathogens classified under the UK High Consequence Infectious Diseases (HCID) framework, we previously evaluated both a whole-genome tiling amplicon sequencing scheme and a pan-viral hybridisation capture approach (TWIST-CVRP) for sequencing Andes virus (ANDV). In light of the recent outbreak, we make available viral sequencing datasets generated using a historical ANDV isolate (Chile, 1997). In addition, we provide an evaluation of tiling amplicon scheme performance and present recommended primer updates informed by in silico comparison with the recently released outbreak genome. These datasets are intended to support benchmarking, validation, and optimisation of bioinformatic pipelines across the community.

Between December 2019 and January 2020, two patients suspected of having severe yellow fever were admitted to a tertiary healthcare facility in São Paulo, Brazil, presenting with acute hemorrhagic syndrome and neurological alterations; both cases had fatal outcomes. Upon admission, both tested negative for yellow fever viral RNA, and Sabiá virus (SABV), a New World arenavirus, was identified as the causative pathogen. To date, only four humans naturally acquired SABV infections have been confirmed, all fatal and linked to rural settings. We applied next-generation sequencing to generate complete and near-complete genomes from two patients (SP17 and SP19). Existing molecular diagnostics failed to detect SABV; therefore, new molecular tests were developed. Genetic analyses of SP17 and SP19 genomes along with other arenaviruses, revealed that the new cases were genetically diverse, showing 93-98.2% amino acid identity at the NP level among SP17, SP19, and the 1990 reference strain (SPH114202). Time-scaled phylogenetic analyses confirmed that SP17 and SP19 were not epidemiologically linked and suggested that SABV has been circulating undetected in Brazil for over a century. Additionally, homology modeling and structure-based mapping provided insights into SABV receptor-binding sequence conservation, suggesting that SABV shares similar receptor binding structure to other clade B arenaviruses, despite some amino acid variation around receptor binding site. Our findings underscore the need for retrospective and prospective surveillance of undiagnosed hemorrhagic fever cases to assess the public health impact of SABV in Brazil.

Background:The COVID-19 pandemic exposed critical vulnerabilities in global laboratory supply chains, disrupting the availability of key reagents and jeopardising the continuity of genomic surveillance for epidemic response. Sustaining sequencing capacity during shortages requires locally accessible alternatives to commercial kits. Methods:We developed ARTIC HELP (Homebrew Enzymes for Library Preparation), an open-source adaptation of the ARTIC nanopore sequencing protocol for viral genomic surveillance. We described cost-effective, generic replacements for enzyme mixes used in tiling multiplex RT-PCR and the nanopore native barcoding workflow, including end-prep (EP), barcode ligation (BL), and adapter ligation (AL). Through systematic evaluation, we tested wild-type M-MLV reverse transcriptase and two types of proofreading DNA polymerases, (i) B-family Pfu-based polymerases fused to an Sso7d DNA-binding domain, and (ii) blends of A-family (Taq-based) and B-family (Pfu-based) polymerases, against standard reagents. We validated the workflow on clinical SARS-CoV-2 and Norovirus GII samples. Results:The HELP workflow delivered genome coverage comparable to the ARTIC LoCost protocol. For SARS-CoV-2 samples (Ct ≤28), wild-type M-MLV RT combined with selected Pfu or AB blend polymerases, alongside optimized HELP EP, BL, and AL mixes, achieved 84.0-99.6% genome coverage. For Norovirus GII samples (Ct ≤32), the HELP workflow enabled >85% coverage across six of eight genotypes tested. While some polymerases showed reduced performance at higher Ct values, they performed reliably at Ct <24, supporting their use as emergency alternatives when viral load is high and RNA quality sufficient. Conclusions:ARTIC HELP provides a practical and flexible solution to maintain viral sequencing capacity when standard ARTIC LoCost reagents are inaccessible or unaffordable. Our cost analysis highlights global disparities in reagent pricing, likely influenced by import fees, supply barriers, and local procurement conditions, underscoring the need for more equitable pricing models and local sourcing strategies. By expanding reagent options, ARTIC HELP strengthens preparedness for future global health emergencies.

Measles outbreaks have surged globally in recent years, but current surveillance systems have limited capacity to monitor measles virus (MeV) transmission and evolution at population scale. Although MeV can be detected in wastewater, the public health potential of wastewater genomic surveillance for MeV remains largely unexplored. Here, we deploy sensitive, low-cost MeV wastewater genomic surveillance combining virus concentration, whole-genome amplicon sequencing, and bioinformatic analysis alongside routine clinical genomic surveillance during the 2024-25 outbreak in South Africa. Integrated phylogenetic analyses of wastewater and clinical MeV genomes revealed previously undetected interprovincial spread and transmission links not captured by standard N450 sequencing. Our findings demonstrate that wastewater-integrated whole-genome surveillance expands the coverage and resolution of routine MeV monitoring and provides a scalable tool to advance measles control and elimination efforts. ### Competing Interest Statement KGA has received consulting fees for advising on SARS-CoV-2, variants and the COVID-19 pandemic. The other authors declare no competing interests. ### Funding Statement This work was funded by the Gates Foundation (057213 to KGA, KM, MY; 049272 to KM, MY; 050051 to KM, MY), National Institutes of Health (5T32AI007244-38 to JIL; 3U19AI135995-03S2 to KGA; U19AI135995 to KGA; U01AI151812 to KGA, UL1TR002550 to KGA). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The protocol was reviewed and approved by the University of the Witwatersrand Human Research Ethics Committee (MM220904). The NICD conducts all routine clinical surveillance, including surveillance of notifiable medical conditions, in a protocol reviewed and approved by the University of the Witwatersrand Human Research Ethics Committee (HREC) M210752 and under the legal authority of the National Health Act (no. 61 of 2003). 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. Yes I 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). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All MeV wastewater raw sequencing data and clinical genome sequences produced are available under NCBI BioProject PRJNA1377662.

On 4 September 2025, the Ministry of Public Health, Hygiene and Social Welfare officially declared the 16th Ebola disease outbreak in the Democratic Republic of the Congo (DRC). This outbreak ended on 1 December 2025 and occurred in Bulape Health Zone, Kasaï Province, an area with limited access to appropriate healthcare facilities and resources. Here, we describe the probable index patient and molecular investigations of samples obtained from six suspected patients from the initial outbreak phase. We identified Orthoebolavirus zairense (EBOV) in five samples from different patients. In addition, we performed whole-genome sequencing and generated four complete EBOV genomes. These genomes form a well-supported phylogenetic cluster with genomes from the 1976 Yambuku/Mayinga outbreak. This study suggests a likely new zoonotic spillover event from an as-yet unidentified natural reservoir. While the close relationship to 1976 EBOV Yambuku/Mayinga genomes is striking, this poses additional challenges on the comprehension of the animal reservoir species. The study reports the emergence of a new Ebola variant in a remote area of Kasai Province, DR Congo. The findings highlight the need for rapid detection and sequencing to better identify variants and quickly respond to outbreaks.

Abstract Clinical metagenomics uses sequencing for culture-independent identification of pathogens directly from clinical specimens. While a number of protocols claim to be pathogen agnostic, sensitivity for RNA viruses is likely lower than for bacteria or fungi, as it requires additional processing steps including conversion to cDNA. Sequence-independent, single-primer amplification (SISPA) was first described in 1991, yet how it preferentially enriches viral molecules has never been described. Here we propose that single-primer amplification exploits the PCR suppression effect, which selectively amplifies longer viral molecules over shorter host-derived cDNA fragments on the basis of size. This model predicts that any upstream processing step that disrupts fragment length will prevent this enrichment occurring. To test this, we systematically compared two adapter introduction strategies - during cDNA synthesis and via tagmentation - followed by single primer amplification, using the ZeptoMetrix Respiratory Panel 2.1 containing 16 RNA and 3 DNA virus strains. SISPA-based approaches recovered all of the viral genomes in the control, whereas using tagmentation to amplify cDNA recovered none. We then spiked the controls into extracted clinical samples and found that SISPA-based methods performed best in all background settings, however in high-background settings no viral genomes were recovered by any approach. Finally, using a modified SMART-9N protocol, we demonstrated that single-primer PCR is critical to overall performance, indicating that direct tagmentation of cDNA and dual-primer PCR should be avoided in protocols for clinical metagenomics where high sensitivity for RNA viruses is critical. These findings demonstrate that library preparation strategy fundamentally determines RNA virus sensitivity and offer mechanistic insights for protocol optimisation with direct relevance to clinical metagenomics.

Background The COVID-19 pandemic exposed critical vulnerabilities in global laboratory supply chains, disrupting the availability of key reagents and jeopardising the continuity of genomic surveillance for epidemic response. Sustaining sequencing capacity during shortages requires locally accessible alternatives to commercial kits. Methods We developed ARTIC HELP (Homebrew Enzymes for Library Preparation), an open-source adaptation of the ARTIC nanopore sequencing protocol for viral genomic surveillance. We described cost-effective, generic replacements for enzyme mixes used in tiling multiplex RT-PCR and the nanopore native barcoding workflow, including end-prep (EP), barcode ligation (BL), and adapter ligation (AL). Through systematic evaluation, we tested wild-type M-MLV reverse transcriptase and two types of proofreading DNA polymerases, (i) B-family Pfu-based polymerases fused to an Sso7d DNA-binding domain, and (ii) blends of A-family (Taq-based) and B-family (Pfu-based) polymerases, against standard reagents. We validated the workflow on clinical SARS-CoV-2 and Norovirus GII samples. Results The HELP workflow delivered genome coverage comparable to the ARTIC LoCost protocol. For SARS-CoV-2 samples (Ct ≤28), wild-type M-MLV RT combined with selected Pfu or AB blend polymerases, alongside optimized HELP EP, BL, and AL mixes, achieved 84.0–99.6% genome coverage. For Norovirus GII samples (Ct ≤32), the HELP workflow enabled >85% coverage across six of eight genotypes tested. While some polymerases showed reduced performance at higher Ct values, they performed reliably at Ct <24, supporting their use as emergency alternatives when viral load is high and RNA quality sufficient. Conclusions ARTIC HELP provides a practical and flexible solution to maintain viral sequencing capacity when standard ARTIC LoCost reagents are inaccessible or unaffordable. Our cost analysis highlights global disparities in reagent pricing, likely influenced by import fees, supply barriers, and local procurement conditions, underscoring the need for more equitable pricing models and local sourcing strategies. By expanding reagent options, ARTIC HELP strengthens preparedness for future global health emergencies.

The laboratory contaminant strain Noodlococcus was named for its coccoid cells and unusual colony morphology, which resembled a pile of noodles. Along with laboratory characterisation and electron microscopy, we generated a complete Noodlococcus genome sequence using Illumina and Oxford Nanopore data. The genome consisted of a single, circular, 2,732,108 bp chromosome that shared 97.5% average nucleotide identity (ANI) with the Kocuria rhizophila type strain TA68. We identified genomic features involved in replication (oriC), carotenoid synthesis (crt) and genome defence (CRISPR-Cas) and discovered four novel mobile elements (ISKrh4-7). Despite its environmental ubiquity and relevance to food production, bioremediation and human medicine, there have been few genomic studies of the Kocuria genus. We conducted a comparative, phylogenetic and pangenomic examination of all 257 publicly available Kocuria genomes, with a particular focus on the 56 that were identified as K. rhizophila. We found that there are two phylogenetically distinct clades of K. rhizophila, with within-clade ANI values of 96.7-100.0% and between-clade values of 89.5-90.4%. The second clade, which we refer to as Kocuria pseudorhizophila, exhibited ANI values of <95% relative to TA68 and should constitute a separate species. Delineation of the two clades would be consistent with the rest of the genus, where all other species satisfy the 95% ANI threshold criteria. Differences in the K. rhizophila and K. pseudorhizophila pangenomes likely reflect phenotypic as well as evolutionary divergence. This distinction is relevant to clinical and industrial settings, as strains and genomes from both clades are currently used interchangeably, which may lead to reproducibility issues and phenotype-genotype discordance. Investigating an innocuous laboratory contaminant has therefore provided useful insights into the understudied species K. rhizophila, prompting an unexpected reassessment of its taxonomy.

High numbers of reported mpox cases and recent identification of multiple sustained human outbreaks of mpox virus (MPXV) have highlighted the need for robust, best-practice genomic surveillance tools. In light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, many labs across the globe developed the capacity to do virus genome sequencing; however, MPXV presents additional analytical challenges due to its large genome size, tracts of low-complexity or repeat regions, genetically distinct clades, and the need to perform bespoke apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3)-mutation reconstruction. We present squirrel (Some Quick Reconstruction to Resolve Evolutionary Links), an open source bioinformatic tool that can perform clade-aware alignment, mutation quality assessment, phylogenetic inference, and automated APOBEC3-mutation classification on branches of the phylogeny. Squirrel can be run on the command line or launched through the EPI2ME graphical user interface through the squirrel-nf workflow, enabling robust analysis without need for the command line. With the interactive output report produced and publication-ready APOBEC3-reconstruction visualization, squirrel enables researchers to distinguish between zoonotic and sustained human outbreaks and help accurately inform public health responses.

Influenza A virus poses significant public health challenges, causing seasonal outbreaks and pandemics. Its rapid evolution motivates continuous monitoring of circulating influenza genomes to inform vaccine and antiviral development. Wastewater-based surveillance offers an unbiased, cost-effective approach for genomic surveillance. We developed a novel tiling amplicon primer panel that covers diversity of influenza A virus, targeting segments of the surface proteins HA, NA, and M of subtypes H1N1 and H3N2. Using this panel, we sequenced nucleic acid extracts from 59 Swiss wastewater samples collected at four locations during the 2022/2023 and 2023/2024 winter seasons. We found that wastewater-based abundance estimates of the dominant H1N1 clades correlated with clinical-based estimates in the 2023/2024 season. Furthermore, wastewater-based sequencing revealed mutations in vaccine and drug target sites, consistent with clinical data. Overall, we demonstrate the effectiveness of wastewater-based genomic surveillance of influenza A, including lineage identification and mutation tracking to inform vaccine and antiviral strategies ### Competing Interest Statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Niko Beerenwinkel and Tim R. Julian]report financial support was provided by Swiss National Science Foundation and Swiss Federal Office of Public Health. The other authors, declare to no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ### Funding Statement This study is funded by the Swiss National Science Foundation [Sinergia grant 205933] and the Swiss Federal Office of Public Health (FOPH). S.K. is funded by the Eawag Discretionary Postdoctoral Fellowship. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Ethikkommission Nordwest- und Zentralschweiz confirmed that the research project does not fall under the scope of the Human Research Act, because the project is not defined as a research project as per HRA Art. 2. An authorisation from the ethics committee is therefore not required. 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. Yes I 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). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Digital PCR data of viral concentration of influenza A virus in wastewater are available for download from https://github.com/EawagPHH/RespiratoryVirusesWastewater and wise.ethz.ch. Wastewater sequencing data are available on the European Nucleotide Archive (ENA) under project accession number PRJEB85534. Clinical sequencing data are available on ENA under sample accession numbers SAMEA117552834 and SAMEA117552704. All code used in the analysis and more detailed information about the Bioinformatic analysis is available at https://github.com/cbg-ethz/Influenza\_wastewater\_analysis. [https://github.com/cbg-ethz/Influenza\_wastewater\_analysis][1] [1]: https://github.com/cbg-ethz/Influenza_wastewater_analysis

Hepatitis B virus (HBV) whole genome sequencing (WGS) is currently limited as the DNA viral loads (VL) of many clinical samples are below the threshold required to generate full genomes using current sequencing methods. We developed two pan-genotypic viral enrichment methods, using probe-based capture and tiled amplicon PCR (HEP-TILE) for HBV WGS. We demonstrate using mock samples that both enrichment methods are pan-genotypic (genotypes A-J). Using clinical samples, we demonstrate that HEP-TILE amplification successfully amplifies full genomes at the lowest HBV VL tested (30 IU/ml), and the PCR products can be sequenced using both Nanopore and Illumina platforms. Probe-based capture with Illumina sequencing required VL > 300,000 IU/ml to generate full length HBV genomes. The capture-Illumina and HEP-TILE-Nanopore pipelines had consensus sequencing accuracy of 100% in mock samples with known DNA sequences. Together, these protocols will facilitate the generation of HBV sequence data, enabling a more accurate and representative picture of HBV molecular epidemiology, cast light on persistence and pathogenesis, and enhance understanding of the outcomes of infection and its treatment.

Bacteremia is a serious clinical condition in which pathogenic bacteria enter the bloodstream, putting patients at risk of septic shock and necessitating aggressive antibiotic treatment. Choosing the most effective antibiotic is crucial not only for resolving the infection but also for minimizing side effects, such as dysbiosis in the healthy microbiome and mitigating the evolution of antibiotic resistance. This requires rapid identification of the pathogen and antibiotic susceptibility testing, yet these processes are inherently slow in standard clinical microbiology labs due to reliance on growth-based assays. Although alternative methods exist, they are rarely adopted in clinical settings because they involve complex protocols and high costs for retraining the personnel and new equipment. Here, we present an optimized and straightforward protocol for the rapid and efficient isolation of bacterial pathogens directly from blood samples, without disrupting standard laboratory workflows. This cost-effective approach utilizes commonly available laboratory equipment and enables direct bacterial cell isolation. By eliminating the need for traditional blood culture steps, it significantly reduces diagnostic delays while remaining fully compatible with downstream bacterial identification analyses. Our protocol achieves over 70% bacteria isolation efficiency within 30 min, remained effective at low bacterial concentrations (1–10 bacteria/0.3 mL blood), and preserved bacterial viability with no notable change in growth lag times. We validated the protocol on several clinically relevant bacterial species, including Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. These findings highlight our protocol’s potential utility in clinical and research settings, facilitating timely cultures and minimizing diagnostic delays. Importantly, the ability to rapidly isolate pathogens may offer critical benefits where timely diagnosis directly influences outcomes. For instance, in a neutropenic cancer patient presenting with fever and signs of sepsis, immediate broad-spectrum antibiotics are typically administered empirically. However, without rapid identification of disease causing pathogens, the risk of inappropriate therapy remains high. By enabling pathogen isolation within 30 min, our protocol can facilitate same-day targeted therapy using molecular or spectrometry-based identification methods, improving early treatment decisions, minimizing exposure to ineffective antibiotics, and potentially reducing ICU admissions and mortality.

Since the advent of long-read sequencing, achieving longer read lengths has been a key goal for many users. Ultra-long-read sets (N50 ≥ 100 kb) produced from Oxford Nanopore sequencers have improved genome assemblies in recent years. However, despite progress in extraction protocols and library preparation methods, ultra-long sequencing remains challenging for many sample types. Here, we compare various methods and introduce the FindingNemo protocol that: (1) optimizes ultra-high-molecular-weight (UHMW) DNA extraction and library cleanup by using glass beads and hexamminecobalt(III) chloride (CoHex), (2) can deliver high ultra-long sequencing yield of >20 Gb of reads from a single MinION flow cell or >100 Gb from PromethION devices (R9.4-R10.4 pore variants), and (3) is scalable to using fewer input cells or lower DNA amounts, with extraction to sequencing possible in a single working day. By comparison, we demonstrate that this protocol surpasses previous methods by enabling precise determination of input DNA quantity and quality through cell counting, sample dilution, and homogenization techniques.

Influenza A virus poses significant public health challenges, causing seasonal outbreaks and pandemics. Its rapid evolution motivates continuous monitoring of circulating influenza genomes to inform vaccine and antiviral development. Wastewater-based surveillance offers an unbiased, cost-effective approach for genomic surveillance. We developed a novel tiling amplicon primer panel that covers diversity of influenza A virus, targeting segments of the surface proteins HA, NA, and M of subtypes H1N1 and H3N2. Using this panel, we sequenced nucleic acid extracts from 59 Swiss wastewater samples collected at four locations during the 2022/2023 and 2023/2024 winter seasons. We found that wastewater-based abundance estimates of the dominant H1N1 clades correlated with clinical-based estimates in the 2023/2024 season. Furthermore, wastewater-based sequencing revealed mutations in vaccine and drug target sites, consistent with clinical data. Overall, we demonstrate the effectiveness of wastewater-based genomic surveillance of influenza A, including lineage identification and mutation tracking to inform vaccine and antiviral strategies.

On 4 September 2025, the Ministry of Public Health, Hygiene and Social Welfare officially declared the 16th Ebola virus disease (EVD) outbreak in the Democratic Republic of the Congo (DRC). As of 13 October 2025, the outbreak is ongoing in Bulape Health Zone, Kasaï Province, where access to appropriate healthcare facilities and resources is limited. Here, we describe the probable index patient and molecular investigations of samples obtained from six suspected EVD patients from the initial outbreak phase. We identified Orthoebolavirus zairense (EBOV) in five samples from different patients. In addition, we performed whole-genome sequencing and generated four complete EBOV genomes. These genomes form a well-supported phylogenetic cluster with genomes from the 1976 Yambuku/Mayinga outbreak. This study suggests a likely new zoonotic spillover event from an as-yet unidentified natural reservoir. Bayesian molecular clock analysis estimates the EBOV variants circulating in Bulape, DRC to have emerged from late July to mid-August 2025. While the close relationship to 1976 EBOV Yambuku/Mayinga genomes is striking, this poses additional challenges on the comprehension of the animal reservoir species.

The COVID-19 pandemic exposed vulnerabilities in global laboratory supply chains, disrupting genomic surveillance efforts essential to epidemic response. To address this challenge, we developed ARTIC HELP (Homebrew Enzymes for Library Preparation), a practical, open-source adaptation of the widely adopted ARTIC nanopore sequencing protocol for viral genomic surveillance. We describe generic, cost-effective alternatives to all enzyme mixes used in tiling multiplex RT-PCR amplification of the virus genome, and the nanopore native barcoding workflow, including end-prep (EP), barcode ligation (BL), and adapter ligation (AL), making it broadly applicable to any laboratory. Through systematic evaluation, we identified a wild-type M-MLV reverse transcriptase and two types of proofreading DNA polymerases as effective alternatives when standard reagents are unavailable due to high cost or limited supply: B-family Pfu-based polymerases with a fused Sso7d DNA-binding domain, and blends combining A-family (Taq-based) and B-family (Pfu-based) polymerases. Validation on clinical samples of SARS-CoV-2 and Norovirus GII confirmed that the HELP workflow achieves genome coverage comparable to the ARTIC LoCost protocol. For SARS-CoV-2 samples (Ct ≤28), the wild-type M-MLV RT combined with selected Pfu or A+B polymerases, along with optimised HELP mixes (EP, BL, AL), achieved genome coverage of 84.0–99.6%. For Norovirus GII (Ct ≤32), the HELP workflow using one of the Pfu polymerases achieved genome coverage of >85% for six out of eight genotypes tested. Notably, several of the other polymerases tested showed reduced performance at higher Ct values. However, they still achieved strong coverage at Ct <24, supporting their use as emergency alternatives in rapid outbreak-response sequencing when viral input is high and RNA quality is sufficient. Our approach, ARTIC HELP, provides a framework which can be implemented to address supply chain disruptions, while maintaining robust genomic sequencing capabilities. A cost analysis highlights the well-known significant global disparities in reagent pricing, driven not by protocol differences but by import fees and supply barriers. Thus, our findings highlight the need for fairer global pricing models and support for local sourcing strategies like HELP, to promote equity in genomic research and ensure preparedness for future public health challenges. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, 206298/B/17/Z, 313694/Z/24/Z

This protocol describes the HEP-TILE tiled amplicon protocol for whole genome sequencing of Hepatitis B virus (HBV) on the nanopore MinION. We developed a pan-genotypic (genotypes A-J) HBV scheme using an early version of PrimalScheme3, a web-based primer design tool for developing multiplex primer schemes. PrimalScheme3 is a successor to PrimalScheme, with a number of changes made to enable us to generate an overlapping (tiled) amplicon scheme which covered the circular HBV genome, utilising a number of discrete primers at each position to handle intraspecies diversity. Primer sequences https://github.com/quick-lab/primerschemes/blob/main/primerschemes/hbv/600/v2.1.0/primer.bed The amplicons can also be fragmented and sequenced on Illumina platforms. Recommended extraction protocol We use the QIAamp minelute virus spin kit with carrier RNA. For samples with VL >5log IU/ml we extract from 200ul of sample using the manufacturers protocol, for samples with VL <5log IU/ml we extract from 400ul sample doubling up the protease, AL and ethanol.

This procedure provides instructions on how to generate amplicons and NGS data for the near whole genome of Mpox. Illumina, Oxford Nanopore or other NGS sequencing platforms can all be utilized if amplicon specific library preparations and platform specific bioinformatic analysis pipelines are available. We detail specific use of a modified Illumina DNA prep library preparation and the analysis pipelines using the generated data in this protocol. The BCCDC / ARTIC V2.3.4 Mpox WGS 2500bp primer scheme was optimized to match and efficiently generate data from the global 2022 West African clade outbreak. The MPV_3000_1_Left to MPV_3000_79_Right primers tile the near whole genome of the Mpox virus. Since the first and last ~6500bp of the Mpox genome (ITR) are usually identical, the last ~6500bp of the genome is masked out on the reference genome to simplify analysis using short read Illumina data. The resulting ITR regions thus represent a consensus ITR combining the amplification products from the dual ITR primer binding sites. For specific ITR region sequence information the use of unique external primer binding sites is required along with removal of all but one common internal ITR binding site to generate specific region spanning PCR products. These products can then be sequenced using Nanopore long read sequencing, see here for some candidate primer combinations (MPV_3000_4_RIGHT, MPV_3000_80_LEFT, and MPV_3000_1_LEFT), to provide specific ITR region sequences. This approach was used to generate the BCCDCmpx2 sequence (available on GISAID, EPI_ISL_13351002) which used both Oxford Nanopore and Illumina data to fully sequence the individual ITR regions and repeat regions refractory to short read technologies alone. The sample PCRs are performed in two pools before being combined for library preparation in order to minimize primer interactions and allow near complete coverage tiling of the Mpox genome. Sequences for the primers and locations on reference genomes can be found in the Materials section tables as well as linked file locations and here. Effort were made to increase sequencing efficiency / genome coverage for a given amount of generated sequence data by performing a number of rounds of primer pair concentration re-balancing in the PCR primer pools (Figure 1.). The primer amounts for V2.3.4 are detailed in this protocol and Materials section.