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Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately \u00a360 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries.<\/ns4:p>","DOI":"10.12688\/wellcomeopenres.15518.2","type":"journal-article","created":{"date-parts":[[2020,5,19]],"date-time":"2020-05-19T11:35:38Z","timestamp":1589888138000},"page":"3","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":40,"title":["Rapid in-country sequencing of whole virus genomes to inform rabies elimination programmes"],"prefix":"10.12688","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9990-6299","authenticated-orcid":false,"given":"Kirstyn","family":"Brunker","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gurdeep","family":"Jaswant","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5754-0556","authenticated-orcid":false,"given":"S.M.","family":"Thumbi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2060-4202","authenticated-orcid":false,"given":"Kennedy","family":"Lushasi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahmed","family":"Lugelo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna M.","family":"Czupryna","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fred","family":"Ade","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3137-2355","authenticated-orcid":false,"given":"Gati","family":"Wambura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veronicah","family":"Chuchu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1579-6596","authenticated-orcid":false,"given":"Rachel","family":"Steenson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chanasa","family":"Ngeleja","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Criselda","family":"Bautista","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9290-5146","authenticated-orcid":false,"given":"Daria L.","family":"Manalo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ma. 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Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,8,5]],"date-time":"2024-08-05T20:54:17Z","timestamp":1722891257000},"score":1,"resource":{"primary":{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,5,19]]},"references-count":84,"URL":"https:\/\/doi.org\/10.12688\/wellcomeopenres.15518.2","relation":{"has-review":[{"id-type":"doi","id":"10.3410\/f.737234207.793578898","asserted-by":"object"}]},"ISSN":["2398-502X"],"issn-type":[{"value":"2398-502X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,5,19]]},"assertion":[{"value":"Indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.16987.r37505, Claude Sabeta, OIE Rabies Reference Laboratory, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort, South Africa, 27 Jan 2020, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v1#referee-response-37505","order":0,"name":"referee-response-37505","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Kirstyn Brunker<\/b>; <i>Posted: 11 May 2020<\/i>; <i>We thank the reviewer for their positive remarks and in our revised manuscript we address each of the minor issues as indicated below. <\/i> <i>We now describe how endemic rabies circulates at low incidence and refer to examples from Latin America where rabies control was largely achieved early this century, but persistent foci have remained a challenge for elimination. <\/i> <i>Archived tissue samples from rabid animals and publicly available sequences are potentially very valuable. Archived samples could be sequenced in bulk and therefore at low cost. Together with existing sequences they can characterize historical RABV diversity in a locality from which to compare to contemporary sequences. This comparison can show whether lineages continue to circulate or have disappeared, perhaps as a result of rabies control efforts. We now explain this in the main text.<\/i> <i>We have clarified that the number of sequenced samples in our study do not represent the surveillance capacity of these countries. These samples were accessible through existing collaborations and time allowed for their processing whilst optimizing our protocols and delivering training workshops. <\/i> <i>In the Philippines, dRIT antibody was prepared at RITM using plasmid antigen from National Institute for Infectious Diseases, Tokyo Japan. Rabbit was used for hyperimmunization, purify and biontinlylate (a paper has been submitted by RITM collaborators for publication of this research). FAT antibody was commercial (Fujirebio). We were able to sequence virus from all positively diagnosed samples (from all locations), aside from the one sample from a cat in the Philippines (mentioned in the text) which was diagnosed positive by dRIT but proved negative during preparation for sequencing (notes from the initial diagnosis had indicated that this was a difficult to diagnose case and it was possibly negative).<\/i> <i>We have corrected typographical errors (trizol units)<\/i> <i>We tested n=11 archived samples but are unable to reference these in the methods as we don\u2019t have unedited gel images. However, details and annotated gel images can be found here: <\/i> <b>https:\/\/doi.org\/10.6084\/m9.figshare.10282865.v1<\/b>. <i> In the text we refer only to samples taken right through to sequencing stage (n=2) prior to field trips.<\/i> <i>Extracted RNA first transcribed with Protoscript II kit and subsequently with Lunascript RT supermix, because of the availability of this kit, its ease of application in the lab and its reduced cost (we use reduced reaction volumes to allow 2.5x the normal recommended reactions per kit).<\/i> <i>We are very keen to disseminate the technology and protocols to as wide an audience as possible- note a protocol has now also been added to protocols.io and is referenced in the paper. We have therefore made our protocols openly available and will update them with any improvements as they are discovered (see protocols.io etc etc). We also emphasize caveats of the method (such as cross-contamination) and how they can be best avoided. We are working broadly with laboratories in East Africa (G. Kour) and in the Philippines (C. Bautista) as part of collaborative PhD studentships. We hope to conduct more training workshops, including one later this year for participants from 3 East African countries and funding permitting we will also conduct further training. For example we are currently carrying out training in Peru and through the ARTIC network have been supporting training in this technology elsewhere, including Brazil and several West African countries.<\/i> <i>We have corrected typographical errors (salivary gland samples) <\/i> <i>In general for sample collection, we advise collection of brainstem with homogenisation and storage in RNA shield. This reagent has the advantage of both inactivating the sample and protecting the RNA for future use. RNA later may aslso be used to preserve RNA but does not fully inactivate the virus. Excellent protocols are available for this (CDC, Blueprint etc) which we refer to. Importantly once collected, samples should be frozen as soon as possible in a secure setting not subject to freeze-thaw cycles.<\/i> <i>Our experience has been that training in bioinformatics is lagging in some LMICs, however we are pleased to see several initiatives aiming to address capacity development in this area. We are involved in the development and testing of publicly available support tools, like lab-on-an-SSD, RAMPART and GLUE, for bioinformatics and data management, and we are consolidating our workflow using Snakemake- a workflow management system to create reproducible and scalable data analyses. This process and accompanying tools will make it easier for laboratory scientists learning these methods and we now mention these in the discussion. <\/i>","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v2#referee-comment-4162","order":1,"name":"referee-comment-4162","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.16987.r37507, Herve Bourhy, Unit Lyssavirus Epidemiology and Neuropathology, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, Paris, France, 03 Feb 2020, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v1#referee-response-37507","order":2,"name":"referee-response-37507","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Kirstyn Brunker<\/b>; <i>Posted: 11 May 2020<\/i>; <i>We are grateful for this positive review and critical insights to improve the manuscript. To address the reviewers concerns we have emphasized the potential that we believe real-time sequencing has to inform policy-decisions and management, whilst also highlighting the further work required to put this into practice. As suggested, we now discuss limitations cautioned by the reviewer. In response, we also provide examples of questions that WGS derived from our MinION protocol would be sufficiently resolved and accurate to address. <\/i> &nbsp; <i>We agree that appropriate tube labelling is a concern but argue that this is so for all genomic studies and not specific to our study. Furthermore, we emphasise that our approach is a multiplex PCR, which involves closed tube amplification using combined pools of primers ( two tubes per sample), not 30+ individual reactions, therefore minimising the risk of cross-contamination and risk of mislabelling when working with many tubes. By including a negative control (processed from cDNA stage) in sequencing runs we are able to monitor the level of cross-contamination and identify high levels (based on read coverage across the sample set and how that compares to the controls) that could impact results. While we did note some reads in negative controls the relative proportion compared to the overall mapped reads in runs was low (median 0.03% of reads in the negative, range 0-1.5%) for sequencing runs that were not part of training exercises. Cross-contamination is a general concern for any type of sequencing and we do agree that it will be increasingly hard to control in field situations. We now emphasize that in an endgame scenario, where samples are increasingly valuable and informative and need to be processed rapidly in-situ (i.e. in the field rather than a clinical biology laboratory), sequencing would likely be conducted individually, i.e. one sample per flow cell. In such cases, cross-contamination would not be expected and higher depth of coverage would address basecall ambiguity and mitigate concerns (hopefully including tube labelling!). Nonetheless we appreciate the reviewers apprehension and have therefore reduced our emphasis on how this approach can address complex epidemiological questions. <\/i> &nbsp; <i>The error rate for nanopore sequences following consensus correction is around 0.3-0.7% (for the sequencing chemistry versions we used), where quality scores are Q30. Although higher than Illumina (~0.1%) corrected nanopore sequences should be accurate enough for many uses aside from perhaps within-host variation (although Nathan Grubaugh\u2019s recent paper suggests that further biases can be corrected to enable characterisation of within-host diversity even with amplicon data). We have also further limited consensus miscalls by masking regions with low depth of coverage. Multiple studies have now demonstrated the utility of nanopore sequences for viral surveillance as noted by the references cited- ebola (Dudas et al), yellow fever (Hill et al), zika (Faria et al), dengue(Kafetzopoulou et al<\/i>) <i> etc and how these sequences can be used for applications such as we have suggested in box 1. We disagree that the nanopore error rate will impact sequences to the extent that they cannot discriminate different lineages or host-associated variants, particularly at whole genome resolution, even with minor consensus errors. Comparisons between Ilumina and nanopore generated sequences on the phylogenetic tree show that they group tightly together and therefore the same conclusion would have been reached regardless of platform. Furthermore, nanopore consensus sequencing accuracy continues to improve and has now reached Q50 with R.10.3 flowcells.<\/i> &nbsp; <i>More specifically, we agree that some questions, such as identification of host adaptation (e.g. emergence in foxes from dogs in Turkey) and the mechanisms underpinning persistence in alternative hosts (see Nardus kudu\/dogs\/foxes) may require ultradeep sequencing (e.g. Marston <\/i>et al <i>). These however are questions that we are struggling to resolve no matter what sequencing approach is used, and are indicative of areas of biology for which we require more fundamental understanding. Since MinION approaches will not be key to breakthroughs in these areas, we have removed them from Box 1. Similarly, socio-ecological processes will be challenging to address, but we argue that epidemiological and contextual data will be a greater limitation than genomic data. <\/i> &nbsp; <i>For those questions where we believe a MinION approach could be valuable, we have edited Box 1 to give examples:<\/i> <i>For host associations, such as vampire bat versus dog rabies in Latin America or mongoose (Kotait et al) vs dog rabies in parts of Southern Africa (Nel et al)<\/i> <i>Lineage distributions in the context of political\/ administrative borders, such as provinces in the Philippines (Tohoma et al), states in North Africa (Talbi et al.) and East Africa (Tanzania and Pemba mainland); and assessing transboundary risk as shown by e.g. Trewby et al) <\/i> <i>Confirming epidemiologically connected areas is a clear question for areas in Latin America now, e.g. Peru\/ Bolivia and a logical and feasible extension from studies by Talbi et al. (localities connected by road networks), Bourhy et al. (connectivity between peri-urban and rural areas), Brunker at al. (connectivity between pastoralist areas)<\/i> <i>Identification of long-distance human-mediated movement of lineages (Thailand paper, rural to urban transmission, TZ examples). <\/i> <i>We argue that resolving complexities in dynamics using genomic data integrated with epidemiological data, such as distinguishing genetic lineages and their association with spatio-temporal patterns, would be possible with nanopore consensus sequences e.g. subtypes reported in Bourhy et al. (where divergence between subtypes was ~0.2-14%) should be possible with MinION data even with minor consensus inaccuracies. <\/i> <i>Discriminate variants to verify freedom from dog rabies or identify emergence of new reservoirs, such as those seen in Latin America (Crab-eating fox, Carnieli et al, primate\/marmoset associated variants, Kotait et al). For example, Carnieli et al found a divergence of ~9% in the G gene between RABV isolates from domestic v wild hosts, variants which nanopore WGS are more than capable of discriminating.<\/i> <i>Generally studies from other pathogens demonstrate this potential (previously mentioned above), and although yet to be achieved for rabies, provide optimism for the future. &nbsp;<\/i> &nbsp;","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v2#referee-comment-4163","order":3,"name":"referee-comment-4163","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.16987.r37504, Christine Carrington, Department of Preclinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St. Augustine, Trinidad and Tobago, 10 Feb 2020, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-3\/v1#referee-response-37504","order":4,"name":"referee-response-37504","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by the Wellcome Trust through a Senior Research Fellowship to KH [207569], a Training Fellowship in Public Health and Tropical Medicine to SMT [110330] and Institutional Strategic Support Fund grants to the University of Glasgow [204820]. KB was supported by a Royal Society of Tropical Medicine and Hygiene Small Grant 2017 [GR000892] and the GCRF\/Scottish Funding Council through a grant awarded to the University of Glasgow [EP\/S51584X\/1]. KL is supported by the DELTAS Africa Initiative [Afrique One-ASPIRE\/DEL-15-008]. Afrique One-ASPIRE is funded by a consortium of donors including the African Academy of Sciences (AAS) Alliance for Accelerating Excellence in Science in Africa (AESA), the New Partnership for Africa\u2019s Development Planning and Coordinating (NEPAD) Agency, the Wellcome Trust [107753] and the UK government. The work at APHA was supported by funding from Defra, the Scottish Government and Welsh Government [SE0431]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":5,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}