An international collaborative effort for infectious disease analyses using MinION

The final speaker in the Field sequencing breakout was Dr. Lucky Ronald Runtuwene, from the University of Tokyo. Lucky, described how the sequencing core lab at the University of Tokyo is well-resourced, with 10 technicians, 4 bioinformaticians, and sufficient capability to sequence 100 genomes per week. However, he pointed out that, while this centralised approach may work in developed countries, instrument costs and the complexities of sample transfer make it unsuitable for developing countries.

Lucky described how in 2016, the University of Tokyo, which was one of the first MinION early access participants, invited researchers from 7 countries to participate in their first MinION summer school in Kashiwa, Japan. This meeting led to the formation of the Global Research Alliances for Infectious Diseases (GRAID) — a collaborative, international effort for infectious disease research using MinION, with the aim of generating new genomic knowledge and facilitating knowledge and technology transfer. This consortium has published four papers to date covering the identification of dengue virus and comprehensive drug resistance identification of malaria parasites. They have also held summer schools in a number of countries, including Thailand, Indonesia, and Kenya.

Lucky then went on to describe some of GRAID’s research collaborations in more detail, starting with the analysis of drug resistance in the malaria parasite Plasmodium falciparum. In this study, the team took advantage of the portability of the MinION to perform sequencing on-site in Indonesia, Vietnam, and Thailand. Using a targeted sequencing approach, nine full-length genes related to drug resistance were analysed across ten clinical research samples (from blood samples and dried-spot blood cards). The SNP data allowed the drug resistance of all samples to be inferred. Specific analysis of the K13 gene mutations — which is associated with artemisinin resistance — in 54 additional samples allowed the identification of a novel, region-specific non-synonymous mutation.

Lucky also shared some data from the GRAID’s research into serotyping dengue virus. For this research, the team used a technique known as loop-mediated isothermal amplification (LAMP) – a low-cost, single tube technique for DNA amplification, which does not require the use of a thermal cycler.  In total, over 200 samples collected from the field in Indonesia, Vietnam, and Thailand were sequenced. The team reported that the method enabled the ‘identification and serotyping of the dengue virus with high sensitivity and specificity’. The overall detection rate was 79%, which Lucky stated was highly dependent on the viral titre.

The GRAID consortium has also developed NanoPipe, an analysis tool for nanopore sequencing data, which enables consortium members and other researchers who are not familiar with bioinformatics to more easily analyse nanopore sequencing data.  The tool provides an intuitive graphical interface for sequence alignment, consensus calling and the identification of polymorphisms.

Closing his talk Lucky outlined some of the additional projects currently being carried out by the consortium, which include HLA typing in severe dengue patients, identification of unknown fever-causing pathogens, and determining the drug resistance pattern in HIV.