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

• DOI: 10.1038/s41598-018-26334-3
• Publication Date: 2018-05-23T10:56:47Z
• Authors: Lucky R. Runtuwene; Josef S. B. Tuda; Arthur E. Mongan; Wojciech Makalowski; Martin C. Frith; Mallika Imwong; Suttipat Srisutham; Lan Anh Nguyen Thi; Nghia Nguyen Tuan; Yuki Eshita; Ryuichiro Maeda; Junya Yamagishi; Yutaka Suzuki
• Keywords: 0301 basic medicine; Genotype; sequence variation; Immunology; Plasmodium falciparum; Drug Resistance; Real-Time Polymerase Chain Reaction; Gene; Article; Computational biology; Antimalarials; Nanopores; 03 medical and health sciences; Biochemistry, Genetics and Molecular Biology; Virology; Parasite hosting; Genetics; Animals; Humans; Plasmodium (life cycle); Parasites; Malaria, Falciparum; RNA Sequencing Data Analysis; Molecular Biology; Biology; Immunological Responses in Aquatic Organisms; Immunology and Microbiology; Pharmacology; FOS: Clinical medicine; Life Sciences; Sequence Analysis, DNA; Thailand; Computer science; Malaria; 3. Good health; World Wide Web; Vietnam; Drug resistance; FOS: Biological sciences; Mutation; Drug

AbstractHere, 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.