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Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections

Lerch, Anita and Koepfli, Cristian and Hofmann, Natalie E. and Messerli, Camilla and Wilcox, Stephen and Kattenberg, Johanna H. and Betuela, Inoni and O'Connor, Liam and Mueller, Ivo and Felger, Ingrid. (2017) Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections. BMC genomics, 18. p. 864.

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Abstract

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.; 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.; 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.; 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.
Faculties and Departments:09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH)
09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Department of Medical Parasitology and Infection Biology > Molecular Diagnostics (Felger)
UniBasel Contributors:Lerch, Anita AL and Hofmann, Natalie and Messerli, Camilla
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:BioMed Central
ISSN:1471-2164
e-ISSN:1471-2164
Note:Publication type according to Uni Basel Research Database: Journal article
Language:English
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Last Modified:08 Dec 2017 10:36
Deposited On:08 Dec 2017 10:36

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