RT Journal Article
SR Electronic
T1 Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 121426
DO 10.1101/121426
A1 Anita Lerch
A1 Cristian Koepfli
A1 Natalie Hofmann
A1 Camilla Messerli
A1 Stephen Wilcox
A1 Johanna H. Kattenberg
A1 Inoni Betuela
A1 Liam O’Connor
A1 Ivo Mueller
A1 Ingrid Felger
YR 2017
UL http://biorxiv.org/content/early/2017/04/03/121426.abstract
AB Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. Such high resolution is needed for molecular monitoring of drug efficacy trials. 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 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.