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- Title
Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome.
- Authors
Tessema, Sofonias K; Hathaway, Nicholas J; Teyssier, Noam B; Murphy, Maxwell; Chen, Anna; Aydemir, Ozkan; Duarte, Elias M; Simone, Wilson; Colborn, James; Saute, Francisco; Crawford, Emily; Aide, Pedro; Bailey, Jeffrey A; Greenhouse, Bryan
- Abstract
<bold>Background: </bold>Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited.<bold>Methods: </bold>A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls.<bold>Results: </bold>Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes.<bold>Conclusions: </bold>The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high-throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.
- Subjects
PROTOZOA; SEQUENCE analysis; MALARIA; HAPLOTYPES; RESEARCH funding
- Publication
Journal of Infectious Diseases, 2022, Vol 225, Issue 7, p1227
- ISSN
0022-1899
- Publication type
journal article
- DOI
10.1093/infdis/jiaa527