Combination random isothermal amplification and nanopore sequencing for rapid identification of the causative agent...

Outbreaks of fever of unknown origin start with nonspecific symptoms and case definition is only slowly developed and adapted, therefore, identifying the causative agent is crucial to ensure suitable treatment and control measures. As an alternative method for Polymerase Chain Reaction in molecular diagnostics diagnostic, metagenomics can be applied to identify the pathogen responsible for the outbreak through sequencing all nucleic acids present in a sample extract. Sequencing data obtained can identify new or variants of known agents.

To develop a rapid and field applicable protocol to allow the identification of the causative agent of an outbreak.

We explored a sequencing protocol relying on multiple displacement isothermal amplification and nanopore sequencing in order to allow the identification of the causative agent in a sample. To develop the procedure, a mock sample consisting of supernatant from Zika virus tissue culture was used.

The procedure took under seven hours including sample preparation and data analysis using an offline BLAST search. In total, 63,678 sequence files covering around 10,000 bases were extracted. BLAST search revealed the presence of Zika virus.

In conclusion, the protocol has potential for point of need sequencing to identify RNA viruses. The whole procedure was operated in a suitcase laboratory. However, the procedure is cooling chain dependent and the cost per sequencing run is still high.