Next-Generation Sequencing and Viral Phylogenetics for outbreak control My VT Phan, DPhil Next Generation Sequencing (NGS) All viral pathogens are encoded by RNA or DNA genomes Their genome sequences provide a specific signature to identify and track transmissions Holmes et al. Nature 2016 Ebola virus genome evolves at a defined rate Virus genome sequence can reveal close virus ancestors and potential sources of the infection Background In December 2013, Ebola virus emerged and spread in Guinea, Liberia, and Sierra Leone, resulting in the largest recorded outbreak of Ebola virus disease in history. http://apps.who.int/ebola/current-situation/ebola-situation-report-30-march-2016 To stop Ebola virus transmission in a population 1. Vaccinate before exposure to the virus Problem: No vaccine at the time 2. Treat with antiviral drugs Problem: No effective drugs to stop EBOV 3. Isolate infected people to prevent transmitting the virus QUARANTINE 4. Identify the source and contacts of virus and QUARANTINE Is this a new virus? Where does it come from? How does it spread? Challenges for deep sequencing Ebola Bottleneck 1 Bottleneck 2 Approvals for shipping Data movement >24 hours Actual sample shipment to transfer raw data from Solved by local Sierra Leone to England sequencing facility Solved by rewriting software, performing assemblies in the tent Sequencing tent PHE diagnostic lab Mateneh Ebola Treatment Centre, Makeni, Sierra Leone Diagnostic labs: • Public Health England labs • European mobile labs • Dutch mobile labs Arias et al. Virus Evolution, 2016, 2 (1) Time-resolved phylogenetic tree of EBOV genomes in Sierra Leone Arias et al. Virus Evolution, 2016, 2 (1) Rapid NGS of Ebola cases to track movement of virus Without an open database of contemporary genomes placement of a genome from a new case is not possible http://virological.org/c/ebolavirus, Andrew Rambaut http://www.nextstrain.org/ebola, Richard Neher, Trevor Bedford Movement to Guinea from Sierra Leone Orange: cases from Liberia Where was patient infected? Sequence virus Patient likely to have been infected in village B Utility of outbreak sequencing July 2015, new virus infections in region free of Ebola for previous 130 days Virus sequenced within 48 hours Clustered with viruses from Freetown Patient had recently travelled from Freetown Excluded: 1. Unknown transmission chain 2. Movement from Guinea 3. New zoonosis Arias et al. Virus Evolution, 2016, 2 (1) Sexual transmission of Ebola virus 29 Aug 2015, a post-mortem swab from case K was positive for EBOV, ie. 50 days after the last confirmed case in this district. 5 additional cases in household Variant changes at specific site Rapid NGS of Ebola cases to track movement of virus Without an open database of contemporary genomes placement of a genome from a new case is not possible http://virological.org/c/ebolavirus, Andrew Rambaut http://ebola.nextflu.org/, Richard Neher, Trevor Bedford Movement to Guinea from Sierra Leone Orange: cases from Liberia Geographical movement of EBOV in West Africa Combined all 1,610 EBOV genomes Dudas et al. Nature 2017 (544) p309-315 & Holmes et al. Nature 2016 Spatial and temporal movements of Ebola virus Dudas et al., Nature 2017 (544), pp309-315 Dudas and Rambaut, virological.org/t/phylogeography-of-2014-2015-ebola-virus-epidemic/199 Summary 1. Utility of NGS in clinical and outbreak settings 2. Generated 554 full genomes: 1/3 of EBOV genomes available 3. Performed rapid sequencing and phylogenetic tracing in 24-72 hours of sample NA receipt. 4. Obtained full Ebola virus genomes from atypical sample types eg semen, breast milk 5. Defined a realistic local platform for future outbreak sequencing and phylogenetic analyses. Acknowledgements University of Cambridge Erasmus MC Ian Goodfellow , Armando Arias, Dutch Mobile Labs Lucy Thorne, Sarah Caddy, Matthew Cotten Jia Lu, Luke Meredith Marion Koopmans University of Makeni WHO Umaru Jah, Raoul Emeric, Dhamari Naidoo Alimamy Tarawalie Sierra Leone MOH, Public Health England, DfID, CDC, EM Labs, Wellcome Trust Sanger Institute Volunteer Clinicians, nurses, scientists Paul Kellam, Simon Watson And many others University of Edinburgh University of Oxford Andrew Rambaut, Gytis Dudas Oliver Pybus, Nuno Faria Comparison of assembled EBOV genomes sequenced by Ion Torrent and by MinION .