Nanopore sequencing of drug-resistance-associated genes in malaria parasites, Plasmodium falciparum

Abstract

Here, we report the application of a portable sequencer, MinION, for genotyping the malaria parasite Plasmodium falciparum. In the present study, an amplicon mixture of nine representative genes causing resistance to anti-malaria drugs is diagnosed. First, we developed the procedure for four laboratory strains (3D7, Dd2, 7G8, and K1), and then applied the developed procedure to ten clinical samples. We sequenced and re-sequenced the samples using the obsolete flow cell R7.3 and the most recent flow cell R9.4. Although the average base-call accuracy of the MinION sequencer was 74.3%, performing >50 reads at a given position improves the accuracy of the SNP call, yielding a precision and recall rate of 0.92 and 0.8, respectively, with flow cell R7.3. These numbers increased significantly with flow cell R9.4, in which the precision and recall are 1 and 0.97, respectively. Based on the SNP information, the drug resistance status in ten clinical samples was inferred. We also analyzed K13 gene mutations from 54 additional clinical samples as a proof of concept. We found that a novel amino-acid changing variation is dominant in this area. In addition, we performed a small population-based analysis using 3 and 5 cases (K13) and 10 and 5 cases (PfCRT) from Thailand and Vietnam, respectively. We identified distinct genotypes from the respective regions. This approach will change the standard methodology for the sequencing diagnosis of malaria parasites, especially in developing countries.

Nanopore sequencing: Review of potential applications in functional genomics

“…The Zika virus outbreak was declared an international public health emergency by the World Health Organization (WHO) in 2016, and the Zika in Brazil Real-time Analysis (ZiBRA) project was established to sequence a thousand genomes from Brazil to monitor the epidemiological information (Faria et al, 2016). Furthermore, Runtuwene and colleagues demonstrated a new MinION application for genotyping analysis of the malaria parasite Plasmodium falciparum in Indonesia (Runtuwene et al, 2018), stressing the potential of the nanopore genotyping approach as an effective and practical alternative for the diagnosis of the malaria parasite. The same research group also developed a genomic surveillance system using the nanopore sequencer for the Ebola outbreak in West Africa (Quick et al, 2016).…”

“…This system takes as little as 15-60 min sequencing after receiving the blood sample, realizing an on-the-spot virus testing without the need for the transportation of hazardous specimens. Furthermore, Runtuwene and colleagues demonstrated a new MinION application for genotyping analysis of the malaria parasite Plasmodium falciparum in Indonesia (Runtuwene et al, 2018), stressing the potential of the nanopore genotyping approach as an effective and practical alternative for the diagnosis of the malaria parasite.…”

Applications of sequencing technology in clinical microbial infection

“…74 Moon et al 75 77 A team led by Justin O'Grady in the UK used Nanopore sequencing technology to rapidly identify bacterial genera and species and bacterial resistance genes in patients with lower respiratory tract infections. 78 Runtuwene et al 79 used Nanopore sequencing to genotype the malaria parasite-Plasmodium falciparum-and infer its drug resistance status. The clinical development and application of Nanopore sequencing technology have become a new milestone in precise pathogen detection.…”

Portable sequencer in the fight against infectious disease

“…Consensus calling using bioinformatics tools can improve accuracy to more than 97% [24,25]. When consensus calling was applied to simultaneously screen for multiple antimalarials resistance, it was clear that north Indonesia, north Vietnam, and southeast Thailand had different mutation patterns in K13 gene [26], the gene related to artemisinin resistance, that were in concordance with a major paper describing the artemisinin-resistance related mutations around the Mekong region [27]. Nevertheless, the mutations conferring resistance to chloroquine were similar in these three regions.…”