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Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance


Date: 13th January 2020 | Source: Nature Microbiology

Authors: Xianding Deng, Asmeeta Achari, Scot Federman, Guixia Yu, Sneha Somasekar, Inês Bártolo, Shigeo Yagi, Placide Mbala-Kingebeni, Jimmy Kapetshi, Steve Ahuka-Mundeke, Jean-Jacques Muyembe-Tamfum, Asim A. Ahmed, Vijay Ganesh, Manasi Tamhankar, Jean L. Patterson, Nicaise Ndembi, Dora Mbanya, Lazare Kaptue, Carole McArthur, José E. Muñoz-Medina, Cesar R. Gonzalez-Bonilla, Susana López, Carlos F. Arias, Shaun Arevalo, Steve Miller, Mars Stone, Michael Busch, Kristina Hsieh, Sharon Messenger, Debra A. Wadford, Mary Rodgers, Gavin Cloherty, Nuno R. Faria, Julien Thézé, Oliver G. Pybus, Zoraima Neto, Joana Morais, Nuno Taveira, John R. Hackett Jr., Charles Y. Chiu.

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.

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