Rapid outbreak identification using point of need nanopore sequencing

Purpose: Polymerase Chain Reaction (PCR) is considered as gold standard tests for molecular detection of pathogens. The PCR relies on the use of known sequences of specific target genes. A false negative PCR result might be obtained in case of new variant of a known pathogen or emerging diseases. Alternatively, Next Generation Sequencing (NGS) can be applied to identify the pathogen responsible for the outbreak through sequencing all nucleic acids existing in the collected sample. In addition, NGS gives insight about the origin and variant of the causative agent. In this study we have established a protocol for rapid identification of the cause of unknown fever as well as the origin of infection. Methods & Materials: A RNA mock sample with fever of unknown origin was tested using a sequencing protocol relying on multiple displacement isothermal amplification and oxford nanopore sequencing. The yielded data were analysed using offline BLAST search data bases to facilitate its implementation at low resource settings. Results: The procedure took around 400 minutes including sample preparation and data analysis. In total, 63,678 sequence files were yielded. After running a local BLAST against a viral genome database, approximately 4% of the reads were identified as ZIKV. The complete ZIKV genome sequence was recovered with 2454 reads with an average read length of 685 bases. The in-depth sequence analysis revealed that it was more related to ZIKV isolated from Senegal. Conclusion: In conclusion, the protocol enables rapid and reliable virus identification and serotyping. The procedure has been conducted with a mobile suitcase laboratory, which is easy to use at the point of need in endemic countries. However, the protocol is still cooling chain dependent and the cost per sequencing run are very high.