to facilitate sequencing of the rna genome of ebov , we developed and tested an approach based on reverse transcription pcr , in which whole virus genomes were amplified in overlapping fragments ( figure 1 , panels a , b ; technical appendix ) .
this approach was first validated in a regular laboratory setting in the rocky mountain laboratories of the national institutes of health ( nih ) by using blood samples from nonhuman primates experimentally inoculated with ebov strain makona - gueckedou - c07 ( 10,11 ) .
this validation showed that sequencing information was obtainable for the complete genome with an average of 7,038 reads at every nucleotide position ( read depth ; technical appendix figure 1 , panel a ) .
we observed no sequence differences when comparing the consensus sequence derived from these data to those obtained by using sanger sequencing ( technical appendix figure 1 , panel b ) .
furthermore , by analyzing linearized plasmid dna of known sequence , we established the accuracy of the minion device as 84% for a single read ( technical appendix figure 1 , panels c , d ) . on the basis of this information , and
the fact that read depth can compensate for miscalled nucleotides in individual reads by piling up reads covering the same region , we determined the theoretical probability for > 1 miscalled base ( tpmb ) in a complete minion - sequenced ebov genome to be < 5% when the read depth is > 33 at all positions ( technical appendix figure 1 , panels e and f ) .
c ) sequencing results showing ebola virus load ( expressed as ct value ) , percentage of the genome with a minimum read depth of > 1 or > 33 , mean read depth , theoretical probability for a miscalled base ( tpmb ) , and genbank accession numbers of complete and nearly complete genomes . brackets at left indicate percentage of ebola virus - positive patient samples below each of the 3 cutoff cycle threshold ( ct ) values used in this study ( ct < 21 , < 24 , < 31 ) .
after having validated this approach , minion devices were taken to the centers for disease control and prevention ( cdc)/nih field laboratory that provided diagnostic support for etus in monrovia , liberia , during august 2014may 2015 .
all equipment and reagents necessary for sequencing could be easily transported as checked luggage by a single person on a commercial carrier . in liberia ,
temperatures in the laboratory area used for sequencing ranged from 28 to 32c , necessitating the use of an improvised heat sink for the devices , which consisted of a metal plate of 30  30 cm ( technical appendix figure 2 , panels a , b ) . under field conditions
, we initially failed to produce complete genomes with high confidence because of problems with pcr yields ( online technical appendix figure 3 , panels a , b ) .
however , by implementing a second pcr step , we circumvented this problem and obtained high quality complete genome sequences for 8 of 9 high - virus load samples ( cycle threshold < 21 ) ( figure 1 , panel c ; technical appendix figure 4 , panel a ) . in lower virus load samples , we could obtain only incomplete genome sequences ; however , even in those samples regions for which sequencing information was available generally showed high read depths ( technical appendix figure 4 , panel b ) , suggesting that further optimization of pcrs might also allow complete coverage for these samples .
furthermore , even incomplete genome sequences can provide valuable information during an outbreak , allowing analysis of individual genes and the tracing of transmission chains ( 12 ) .  
using this updated protocol , we achieve a sustained capacity of 4 full - length genomes per day for a single person conducting the laboratory work using 2 minion devices ( figure 1 , panels a , b ) .
however , with the exception of the first 2 sequencing runs , bioinformatics analysis during this mission was mainly completed after returning to the nih , to maximize the time for raw data acquisition ( online technical appendix ) .
phylogenetic analysis of the complete genomes generated in monrovia , liberia , showed them being clearly distinct from sierra leone or early guinea sequences of ebov - makona ( technical appendix appendix 5 ) but clustering well with all other sequences found in samples from liberia .
these results suggest that ebov in liberia resulted from a single introduction or a limited number of introductions with genetically similar viruses . when analyzing the obtained full - length sequences and comparing them to a consensus sequence from the outbreak ( 13 ) , we observed few mutations , most in noncoding regions or synonymous mutations ( figure 2 ) ; none affected sirna target sequences or the diagnostic targets used in the cdc / nih laboratory .   observed mutations in the 8 fully nanopore - sequenced ebola - positive blood samples compared to a reference sequence from june 2014 ( sli / makona - em106 , genbank accession number km233036.1 ) .
squares indicate nonsynonymous mutations , circles indicate synonymous changes , and triangles indicate changes in noncoding regions .   using bayesian analysis including these sequences , we estimated the nucleotide substitution rate during the outbreak at 1.36  10 , consistent with recently published values ( 5,1315 ) . in a root - to - tip - analysis ,
the sequences we obtained showed substitution rates comparable to other sequences from the outbreak ( online technical appendix figure 6 ) .
overall , these data suggest that ebov has remained relatively stable genetically during the outbreak .
we found that , because of the device s small size and comparatively modest resource requirements , nanopore sequencing has tremendous potential for use in remote and resource - limited areas , and its implementation could revolutionize the capacity of public health professionals to perform sequencing during future disease outbreaks .
although we used a directed approach to sequencing , approaches not dependent on prior pathogen identification ( i.e. for diagnostic use of the minion device ) are currently being developed ( 7 ) and will even further increase this technology s usefulness in future outbreaks .
technical appendix . laboratory procedures , phylogenetic analysis of ebola virus cdna genomes , and bioinformatics scripts used in study of nanopore sequencing as a rapidly deployable ebola outbreak tool .
 rapid sequencing of rna / dna from pathogen samples obtained during disease outbreaks provides critical scientific and public health information . 
 however , challenges exist for exporting samples to laboratories or establishing conventional sequencers in remote outbreak regions . 
 we successfully used a novel , pocket - sized nanopore sequencer at a field diagnostic laboratory in liberia during the current ebola virus outbreak . 
The Study
Conclusions
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