Shaun Arevalo, Jean-Jacques Muyembe-Tamfum, Guixia Yu, Asim A. Ahmed, Steve Ahuka-Mundeke, Mary A. Rodgers, Mars Stone, César González-Bonilla, Nuno R. Faria, Carole McArthur, Xianding Deng, Charles Y. Chiu, Lazare Kaptue, Zoraima Neto, Jean L. Patterson, Vijay S. Ganesh, Sneha Somasekar, Kristina Hsieh, Carlos F. Arias, Jimmy Kapetshi, Manasi Tamhankar, Nuno Taveira, Asmeeta Achari, Oliver G. Pybus, Placide Mbala-Kingebeni, Scot Federman, Shigeo Yagi, Sharon Messenger, Inês Bártolo, Nicaise Ndembi, Michael P. Busch, Debra A. Wadford, Steve Miller, Dora Mbanya, John R. Hackett, Susana López, José Esteban Muñoz-Medina, Gavin Cloherty, Joana Morais, Julien Thézé, University of Oxford [Oxford], and Abbott Laboratories Appeared in article as Abbott Laboratories NIH from the NIAID R33-AI129455 United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Heart Lung & Blood Institute (NHLBI) Appeared in article as NIH from the National Heart, Lung, and Blood Institute R01-HL105704 California Initiative to Advance Precision Medicine Charles and Helen Schwab Foundation Steven and Alexandra Cohen Foundation United States Department of Defense Appeared in article as United States Department of Defense W81XWH-17-1-0681 Wellcome Trust Appeared in article as Wellcome Trust Royal Society/Sir Henry Dale Fellowship 204311/Z/16/Z Global Challenges Research Fund 005073 Oxford John Fell Research Fund 005166 Africa Oxford grant AfiOx-48
Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use., This study describes a new method that improves the sensitivity of viral detection compared with next-generation sequencing and enables the detection of emerging flaviviruses not specifically targeted a priori. Metagenomic sequencing with spiked primer enrichment is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making it applicable for diagnostic laboratory and field use.