Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2017
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
_version_ 1782505596961947648
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
work_keys_str_mv AT cowellannien selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT loydorothye selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT sundararamansesha selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT valdiviahugo selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT fischkathleen selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT lescanoandresg selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT baldevianogchristian selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT durandsalomon selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT gerbasivince selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT sutherlandcolinj selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT nolderdebbie selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT vinetzjosephm selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT hahnbeatriceh selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples
AT winzelerelizabetha selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofplasmodiumvivaxfromunprocessedclinicalsamples