Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections
Web of Science
AuthorLerch, A; Koepfli, C; Hofmann, NE; Messerli, C; Wilcox, S; Kattenberg, JH; Betuela, I; O'Connor, L; Mueller, I; Felger, I
Source TitleBMC Genomics
AffiliationMedical Biology (W.E.H.I.)
Medicine Dentistry & Health Sciences
Document TypeJournal Article
CitationsLerch, A., Koepfli, C., Hofmann, N. E., Messerli, C., Wilcox, S., Kattenberg, J. H., Betuela, I., O'Connor, L., Mueller, I. & Felger, I. (2017). Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections. BMC GENOMICS, 18 (1), https://doi.org/10.1186/s12864-017-4260-y.
Access StatusOpen Access
BACKGROUND: Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum. METHODS: Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software "HaplotypR" was developed for data analysis. RESULTS: Cpmp was highly diverse (He = 0.96) in contrast to csp (He = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold. CONCLUSIONS: To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10'000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.
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