Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of<italic toggle="yes"> Plasmodium vivax</italic> from Unprocessed Clinical Samples
ABSTRACT 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...
Guardado en:
| Autores principales: | , , , , , , , , , , , , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
American Society for Microbiology
2017
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/06471cd2d0064efd87efc3184399387a |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| id |
oai:doaj.org-article:06471cd2d0064efd87efc3184399387a |
|---|---|
| record_format |
dspace |
| spelling |
oai:doaj.org-article:06471cd2d0064efd87efc3184399387a2021-11-15T15:51:07ZSelective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of<italic toggle="yes"> Plasmodium vivax</italic> from Unprocessed Clinical Samples10.1128/mBio.02257-162150-7511https://doaj.org/article/06471cd2d0064efd87efc3184399387a2017-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02257-16https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.Annie N. CowellDorothy E. LoySesh A. SundararamanHugo ValdiviaKathleen FischAndres G. LescanoG. Christian BaldevianoSalomon DurandVince GerbasiColin J. SutherlandDebbie NolderJoseph M. VinetzBeatrice H. HahnElizabeth A. WinzelerAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 8, Iss 1 (2017) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
Microbiology QR1-502 |
| spellingShingle |
Microbiology QR1-502 Annie N. Cowell Dorothy E. Loy Sesh A. Sundararaman Hugo Valdivia Kathleen Fisch Andres G. Lescano G. Christian Baldeviano Salomon Durand Vince Gerbasi Colin J. Sutherland Debbie Nolder Joseph M. Vinetz Beatrice H. Hahn Elizabeth A. Winzeler Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of<italic toggle="yes"> Plasmodium vivax</italic> from Unprocessed Clinical Samples |
| description |
ABSTRACT 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. IMPORTANCE Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS. |
| format |
article |
| author |
Annie N. Cowell Dorothy E. Loy Sesh A. Sundararaman Hugo Valdivia Kathleen Fisch Andres G. Lescano G. Christian Baldeviano Salomon Durand Vince Gerbasi Colin J. Sutherland Debbie Nolder Joseph M. Vinetz Beatrice H. Hahn Elizabeth A. Winzeler |
| author_facet |
Annie N. Cowell Dorothy E. Loy Sesh A. Sundararaman Hugo Valdivia Kathleen Fisch Andres G. Lescano G. Christian Baldeviano Salomon Durand Vince Gerbasi Colin J. Sutherland Debbie Nolder Joseph M. Vinetz Beatrice H. Hahn Elizabeth A. Winzeler |
| author_sort |
Annie N. Cowell |
| title |
Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of<italic toggle="yes"> Plasmodium vivax</italic> from Unprocessed Clinical Samples |
| title_short |
Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of<italic toggle="yes"> Plasmodium vivax</italic> from Unprocessed Clinical Samples |
| title_full |
Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of<italic toggle="yes"> Plasmodium vivax</italic> from Unprocessed Clinical Samples |
| title_fullStr |
Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of<italic toggle="yes"> Plasmodium vivax</italic> from Unprocessed Clinical Samples |
| title_full_unstemmed |
Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of<italic toggle="yes"> Plasmodium vivax</italic> from Unprocessed Clinical Samples |
| title_sort |
selective whole-genome amplification is a robust method that enables scalable whole-genome sequencing of<italic toggle="yes"> plasmodium vivax</italic> from unprocessed clinical samples |
| publisher |
American Society for Microbiology |
| publishDate |
2017 |
| url |
https://doaj.org/article/06471cd2d0064efd87efc3184399387a |
| work_keys_str_mv |
AT anniencowell selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT dorothyeloy selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT seshasundararaman selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT hugovaldivia selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT kathleenfisch selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT andresglescano selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT gchristianbaldeviano selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT salomondurand selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT vincegerbasi selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT colinjsutherland selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT debbienolder selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT josephmvinetz selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT beatricehhahn selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples AT elizabethawinzeler selectivewholegenomeamplificationisarobustmethodthatenablesscalablewholegenomesequencingofitalictoggleyesplasmodiumvivaxitalicfromunprocessedclinicalsamples |
| _version_ |
1718427379417743360 |