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

Anita Lerch, Cristian Koepfli, Natalie E Hofmann, Camilla Messerli, Stephen Wilcox, Johanna H Kattenberg, Inoni Betuela, Liam O'Connor, Ivo Mueller, Ingrid Felger

Research output: Contribution to journalA1: Web of Science-article

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Abstract

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.

Original languageEnglish
JournalBMC Genomics
Volume18
Pages (from-to)864
Number of pages13
ISSN1471-2164
DOIs
Publication statusPublished - 2017

Keywords

  • Journal Article

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