Nanopore sequencing for genotyping pathogens of tropical diseases

Nanopore sequencer, MinION, has enabled sequencing analysis without pre-installation of expensive conventional sequencers or pre-requisite of specific skills in biological experiments. Even electric supply is not always necessary, by connecting MinION to a laptop PC. These features of MinION have opened the opportunity to enable precise genotyping of pathogens in tropical diseases in a developing country even in its filed areas. In this study, we attempted genotyping Dengue viruses regarding their serotypes (types 1-4). We directly used serum samples of Indonesian Dengue patients, from which viral genomes were directly amplified by the reverse-transcription-LAMP method in an isothermal reaction condition. We directly used the amplified templates for MinION sequencing allocating one flow cell per sample. We found, although the overall sequencing quality was low (70% sequence identify to the reference genome and the quality value of QV= 5 on average), thereby obtained sequence data could discriminate different serotypes of the viruses, whose genome sequences were diverged with the sequence similarity of 70%, with the overall accuracy of 98%. To further examine whether MinION sequencing can be also applied for detecting SNVs, we conducted genotyping of presumed drug resistance-causing SNVs in malaria parasites, Plasmodium falciparum. We similarly subjected ten PCR amplicon-mixes covering these SNVs to the MinION sequencing. In spite of the fact that the sequence alignments generated by a Smith-Waterman-based program, SSEACH showed that the average sequence identity was 65%, we found that the mutations at a particular position could be called by the accuracy of 85%, when all the reads covering the corresponding positions were collectively evaluated. Taken together, we provide the first simple experimental and analytical MinION sequencing procedure, which can be easily followed in a developing country to effectively genotype pathogens of tropical diseases.