Sequencing of virus genomes during disease outbreaks can provide valuable information for diagnostics, epidemiology, and evaluation of potential countermeasures. However, particularly in remote areas logistical and technical challenges can be significant. Nanopore sequencing provides an alternative to classical Sanger and next-generation sequencing methods, and was successfully used under outbreak conditions (Hoenen et al., 2016; Quick et al., 2016). Here we describe a protocol used for sequencing of Ebola virus under outbreak conditions using Nanopore technology, which we successfully implemented at the CDC/NIH diagnostic laboratory (de Wit et al., 2016) located at the ELWA-3 Ebola virus Treatment Unit in Monrovia, Liberia, during the recent Ebola virus outbreak in West Africa.
[Background] Determining the full-length sequence of virus genomes is an essential procedure in virology. While the classical approach to this involved Sanger sequencing following a primer-walking strategy, newer approaches involve the use of next-generation sequencing methods such as 454, Illumina or Ion Torrent technologies. A common problem with all these technologies, despite their many advantages, is that the required instrumentation is large, expensive, fragile, and therefore difficult to transport. Also, library preparation procedures are often involved. While under usual circumstances these issues are of little consequence, as these machines are run in specialized laboratories with excellent infrastructure, during virus outbreaks in remote areas (for example in case of ebolavirus outbreaks) this can pose significant problems, particularly since the export of samples from affected areas to these specialized laboratories is often politically and logistically challenging. Under these circumstances, the availability of a sequencing technology that can be easily and quickly deployed into remote areas, and allows sequencing to be done directly in an outbreak area, can be invaluable. Therefore, we have tested the MinION sequencing device, which at that time was under development by Oxford Nanopore Technologies (ONT), at the field diagnostic laboratory at the ELWA-3 Ebola virus Treatment Unit in Monrovia (de Wit et al., 2016) during the recent Ebola virus outbreak in West Africa, and developed a protocol for the rapid generation of full-length sequences of Ebola viruses under these conditions. This device employs nanopores, through which nucleotide-strands are transported in a controlled fashion. The nucleotides block and thus modulate an ion-current flowing through those pores, depending on the physical properties of the nucleotides passing though the nanopores, and these current modulations are measured by the device and translated into nucleotide sequences. Results of this test, which indicated that this technology indeed shows great promise as a rapidly deployable and highly usable sequencing platform, are available elsewhere (Hoenen et al., 2016), as are the results of a similar test using the same sequencing platform performed independently of our own efforts by Quick et al. (2016).
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