Newcastle by analyzing linearized plasmid DNA of

Newcastle Disease outbreaks pose a
great challenge to outbreak responses. Rapidly obtaining genetic information to
differentiate NDV genotypes during disease outbreaks is crucial for virus
evolution, and monitoring the validity of diagnostic assays. Further, rapid
results may help determine the efficacy of sequence-dependent countermeasures
including vaccination strategies. Such work depends on rapid sequencing of viral material directly from
clinical samples—i.e., without the need to isolate the virus in pure culture. We
established a protocol and evaluated the feasibility of Nanopore sequencing
technology using a MinION sequencing device (Oxford Nanopore Technologies. This
is a cost effective, rapid, and can differentiate all representative genotypes
of APMV-1 viruses. To facilitate sequencing and
identification of the RNA genome of APMV-1, we developed and tested an approach
based on reverse transcription PCR, in which Matrix/Fusion gene region was
amplified. This approach was first validated on using egg grown viruses and
then clinical samples collected from field conditions during disease outbreaks
in Pakistan from chicken were used for its clinical applicability. This
validation showed that sequencing information was obtainable with an average of
7,038 reads at every nucleotide position. We observed no sequence differences
when comparing the consensus sequence derived from these data to those obtained
by using Miseq and between consecutive runs of same samples on MinION.
Furthermore, by analyzing linearized plasmid DNA of known sequence, we
established the accuracy of the MinION device as 100 % for a consensus
sequence. 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. 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 and will even further
increase this technology’s usefulness in future outbreaks. Further, data
turnaround is very rapid, and consequently, nanopore sequencing is being
developed as a rapid diagnostic tool for management of outbreaks of various