A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples
Abstract Background VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathoge...
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ftdoajarticles:oai:doaj.org/article:ec4372aa93a44f96a4e911ed8b3f84e6 2023-05-15T15:16:51+02:00 A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples Antoine Dara Mark A. Travassos Matthew Adams Sarah Schaffer DeRoo Elliott F. Drábek Sonia Agrawal Miriam K. Laufer Christopher V. Plowe Joana C. Silva 2017-08-01T00:00:00Z https://doi.org/10.1186/s12936-017-1976-8 https://doaj.org/article/ec4372aa93a44f96a4e911ed8b3f84e6 EN eng BMC http://link.springer.com/article/10.1186/s12936-017-1976-8 https://doaj.org/toc/1475-2875 doi:10.1186/s12936-017-1976-8 1475-2875 https://doaj.org/article/ec4372aa93a44f96a4e911ed8b3f84e6 Malaria Journal, Vol 16, Iss 1, Pp 1-9 (2017) var2csa Sequencing PacBio Malaria Vaccines Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 article 2017 ftdoajarticles https://doi.org/10.1186/s12936-017-1976-8 2022-12-31T14:43:31Z Abstract Background VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues. Methods PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module. Results Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus. Conclusions This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Pacific Malaria Journal 16 1 |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
var2csa Sequencing PacBio Malaria Vaccines Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
spellingShingle |
var2csa Sequencing PacBio Malaria Vaccines Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 Antoine Dara Mark A. Travassos Matthew Adams Sarah Schaffer DeRoo Elliott F. Drábek Sonia Agrawal Miriam K. Laufer Christopher V. Plowe Joana C. Silva A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples |
topic_facet |
var2csa Sequencing PacBio Malaria Vaccines Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
description |
Abstract Background VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues. Methods PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module. Results Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus. Conclusions This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other ... |
format |
Article in Journal/Newspaper |
author |
Antoine Dara Mark A. Travassos Matthew Adams Sarah Schaffer DeRoo Elliott F. Drábek Sonia Agrawal Miriam K. Laufer Christopher V. Plowe Joana C. Silva |
author_facet |
Antoine Dara Mark A. Travassos Matthew Adams Sarah Schaffer DeRoo Elliott F. Drábek Sonia Agrawal Miriam K. Laufer Christopher V. Plowe Joana C. Silva |
author_sort |
Antoine Dara |
title |
A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples |
title_short |
A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples |
title_full |
A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples |
title_fullStr |
A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples |
title_full_unstemmed |
A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples |
title_sort |
new method for sequencing the hypervariable plasmodium falciparum gene var2csa from clinical samples |
publisher |
BMC |
publishDate |
2017 |
url |
https://doi.org/10.1186/s12936-017-1976-8 https://doaj.org/article/ec4372aa93a44f96a4e911ed8b3f84e6 |
geographic |
Arctic Pacific |
geographic_facet |
Arctic Pacific |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Malaria Journal, Vol 16, Iss 1, Pp 1-9 (2017) |
op_relation |
http://link.springer.com/article/10.1186/s12936-017-1976-8 https://doaj.org/toc/1475-2875 doi:10.1186/s12936-017-1976-8 1475-2875 https://doaj.org/article/ec4372aa93a44f96a4e911ed8b3f84e6 |
op_doi |
https://doi.org/10.1186/s12936-017-1976-8 |
container_title |
Malaria Journal |
container_volume |
16 |
container_issue |
1 |
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1766347141487263744 |