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Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH
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A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples.

Antoine Dara1, Mark A Travassos1, Matthew Adams1

  • 1Division of Malaria Research, Institute for Global Health, University of Maryland School of Medicine, Baltimore, MD, USA.

Malaria Journal
|August 19, 2017
PubMed
Summary

Pacific Biosciences (PacBio) sequencing enables efficient recovery and genetic variation analysis of the polymorphic VAR2CSA gene from Plasmodium falciparum, crucial for placental malaria vaccine development.

Keywords:
MalariaPacBioSequencingVaccinesvar2csa

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Area of Science:

  • Genomics
  • Parasitology
  • Vaccine Development

Background:

  • VAR2CSA, a Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), is key in placental malaria pathogenesis and a leading vaccine candidate.
  • VAR2CSA's high polymorphism and the limitations of traditional sequencing methods (capillary electrophoresis) hinder comprehensive genetic analysis, especially from polyclonal clinical samples.

Purpose of the Study:

  • To develop and validate a next-generation sequencing approach using Pacific Biosciences (PacBio) for sequencing the VAR2CSA gene.
  • To overcome the challenges associated with sequencing polymorphic genes like VAR2CSA from clinical samples.

Main Methods:

  • Designed PCR primers targeting a 5 kb segment of the 5' end of the var2csa gene.
  • Sequenced PCR amplicons using PacBio sequencing technology.
  • Validated the method on DNA from clinical samples, including cryopreserved erythrocytes and dried blood spots.

Main Results:

  • Successfully recovered a ~5 kb segment encoding the majority of VAR2CSA's extracellular region from clinical samples.
  • Demonstrated high success rates with cryopreserved samples and moderate success with dried blood spots.
  • Characterized the genetic variation within the var2csa locus.

Conclusions:

  • The PacBio sequencing approach provides a feasible and efficient method for analyzing var2csa genetic variation.
  • This method can be adapted to sequence other highly variable Plasmodium falciparum genes, aiding in vaccine research and diagnostics.