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Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples
Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challengi...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296604/ https://www.ncbi.nlm.nih.gov/pubmed/28174312 http://dx.doi.org/10.1128/mBio.02257-16 |
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author | Cowell, Annie N. Loy, Dorothy E. Sundararaman, Sesh A. Valdivia, Hugo Fisch, Kathleen Lescano, Andres G. Baldeviano, G. Christian Durand, Salomon Gerbasi, Vince Sutherland, Colin J. Nolder, Debbie Vinetz, Joseph M. Hahn, Beatrice H. Winzeler, Elizabeth A. |
author_facet | Cowell, Annie N. Loy, Dorothy E. Sundararaman, Sesh A. Valdivia, Hugo Fisch, Kathleen Lescano, Andres G. Baldeviano, G. Christian Durand, Salomon Gerbasi, Vince Sutherland, Colin J. Nolder, Debbie Vinetz, Joseph M. Hahn, Beatrice H. Winzeler, Elizabeth A. |
author_sort | Cowell, Annie N. |
collection | PubMed |
description | Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. |
format | Online Article Text |
id | pubmed-5296604 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-52966042017-02-13 Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples Cowell, Annie N. Loy, Dorothy E. Sundararaman, Sesh A. Valdivia, Hugo Fisch, Kathleen Lescano, Andres G. Baldeviano, G. Christian Durand, Salomon Gerbasi, Vince Sutherland, Colin J. Nolder, Debbie Vinetz, Joseph M. Hahn, Beatrice H. Winzeler, Elizabeth A. mBio Research Article Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. American Society for Microbiology 2017-02-07 /pmc/articles/PMC5296604/ /pubmed/28174312 http://dx.doi.org/10.1128/mBio.02257-16 Text en Copyright © 2017 Cowell et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Research Article Cowell, Annie N. Loy, Dorothy E. Sundararaman, Sesh A. Valdivia, Hugo Fisch, Kathleen Lescano, Andres G. Baldeviano, G. Christian Durand, Salomon Gerbasi, Vince Sutherland, Colin J. Nolder, Debbie Vinetz, Joseph M. Hahn, Beatrice H. Winzeler, Elizabeth A. Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples |
title | Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples |
title_full | Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples |
title_fullStr | Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples |
title_full_unstemmed | Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples |
title_short | Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples |
title_sort | selective whole-genome amplification is a robust method that enables scalable whole-genome sequencing of plasmodium vivax from unprocessed clinical samples |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296604/ https://www.ncbi.nlm.nih.gov/pubmed/28174312 http://dx.doi.org/10.1128/mBio.02257-16 |
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