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Development and Optimization of a Selective Whole-Genome Amplification To Study Plasmodium ovale Spp.

V Joste1,2,3, E Guillochon1, J Clain1

  • 1Université Paris Cité, IRD, MERIT, Paris, France.

Microbiology Spectrum
|September 13, 2022
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Summary

New selective whole-genome amplification (sWGA) protocols enable high-quality whole-genome sequencing (WGS) for Plasmodium ovale parasites. This advances population genomic studies of P. ovale species, crucial for understanding malaria relapse and virulence.

Keywords:
Mcrbc endonucleasePlasmodium ovale spp.dihydrofolate reductaseorthologssWGAwhole genome

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Area of Science:

  • Malariology
  • Genomics
  • Parasitology

Background:

  • Plasmodium ovale spp. comprises two species, P. ovale wallikeri and P. curtisi, important human malaria parasites.
  • Whole-genome sequencing (WGS) offers insights into P. ovale spp. evolution but is hindered by low parasitemia, human DNA contamination, and lack of in vitro culture.
  • Understanding P. ovale spp. relapse and virulence requires high-quality genomic data.

Purpose of the Study:

  • To develop and validate selective whole-genome amplification (sWGA) protocols for P. ovale spp. whole-genome sequencing (WGS).
  • To optimize sWGA protocols using genomic DNA preincubation with McrBC endonuclease.
  • To enable large-scale population genomic studies of P. ovale spp.

Main Methods:

  • Developed two sWGA protocols tested on clinical samples of P. ovale wallikeri and P. ovale curtisi.
  • Utilized blood leukodepletion by cellulose-based filtration as a gold standard for comparison.
  • Incorporated genomic DNA preincubation with McrBC endonuclease to enhance amplification efficiency.

Main Results:

  • Obtained high-quality WGS data with >80% genome coverage (≥5 reads) for all samples.
  • Identified over 5,000 unique single-nucleotide polymorphisms (SNPs) per species.
  • Detected amino acid changes in pocdhfr and powdhfr associated with drug resistance.

Conclusions:

  • The developed sWGA protocols are effective for generating high-quality WGS data from P. ovale spp.
  • These protocols overcome challenges associated with low parasitemia and human DNA contamination.
  • The new methods will facilitate crucial large-scale population genomic studies of P. ovale spp.