Dataset Information

Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax 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 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.

SUBMITTER: Cowell AN

PROVIDER: S-EPMC5296604 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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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.

Cowell Annie N AN Loy Dorothy E DE Sundararaman Sesh A SA Valdivia Hugo H Fisch Kathleen K Lescano Andres G AG Baldeviano G Christian GC Durand Salomon S Gerbasi Vince V Sutherland Colin J CJ Nolder Debbie D Vinetz Joseph M JM Hahn Beatrice H BH Winzeler Elizabeth A EA

mBio 20170207 1

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 difficul ...[more]

PMID: 28174312

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Project description:Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections causing significant morbidity and mortality among children and the elderly; two RSV vaccines and a monoclonal antibody have recently been approved. Thus, there is an increasing need for a detailed and continuous genomic surveillance of RSV circulating in resource-rich and resource-limited settings worldwide. However, robust, cost-effective methods for whole genome sequencing of RSV from clinical samples that are amenable to high-throughput are still scarce. We developed Next-RSV-SEQ, an experimental and computational pipeline to generate whole genome sequences of historic and current RSV genotypes by in-solution hybridization capture-based next generation sequencing. We optimized this workflow by automating library preparation and pooling libraries prior to enrichment in order to reduce hands-on time and cost, thereby augmenting scalability. Next-RSV-SEQ yielded near-complete to complete genome sequences for 98% of specimens with Cp values ≤31, at median on-target reads >93%, and mean coverage depths between ~1,000 and >5,000, depending on viral load. Whole genomes were successfully recovered from samples with viral loads as low as 230 copies per microliter RNA. We demonstrate that the method can be expanded to other respiratory viruses like parainfluenza virus and human metapneumovirus. Next-RSV-SEQ produces high-quality RSV genomes directly from culture isolates and, more importantly, clinical specimens of all genotypes in circulation. It is cost-efficient, scalable, and can be extended to other respiratory viruses, thereby opening new perspectives for a future effective and broad genomic surveillance of respiratory viruses.ImportanceRespiratory syncytial virus (RSV) is a leading cause of severe acute respiratory tract infections in children and the elderly, and its prevention has become an increasing priority. Recently, vaccines and a long-acting monoclonal antibody to protect effectively against severe disease have been approved for the first time. Hence, there is an urgent need for genomic surveillance of RSV at the global scale to monitor virus evolution, especially with an eye toward immune evasion. However, robust, cost-effective methods for RSV whole genome sequencing that are suitable for high-throughput of clinical samples are currently scarce. Therefore, we have developed Next-RSV-SEQ, an experimental and computational pipeline that produces reliably high-quality RSV genomes directly from clinical specimens and isolates.

Project description:The genomic diversity of Plasmodium malariae malaria parasites is understudied, partly because infected individuals tend to present with low parasite densities, leading to difficulties in obtaining sufficient parasite DNA for genome analysis. Selective whole genome amplification (SWGA) increases the relative levels of pathogen DNA in a clinical sample, but has not been adapted for P. malariae parasites. Here we design customized SWGA primers which successfully amplify P. malariae DNA extracted directly from unprocessed clinical blood samples obtained from patients with P. malariae-mono-infections from six countries, and further test the efficacy of SWGA on mixed infections with other Plasmodium spp. SWGA enables the successful whole genome sequencing of samples with low parasite density (i.e. one sample with a parasitaemia of 0.0064% resulted in 44% of the genome covered by ≥ 5 reads), leading to an average 14-fold increase in genome coverage when compared to unamplified samples. We identify a total of 868,476 genome-wide SNPs, of which 194,709 are unique across 18 high-quality isolates. After exclusion of the hypervariable subtelomeric regions, a high-quality core subset of 29,899 unique SNPs is defined. Population genetic analysis suggests that P. malariae parasites display clear geographical separation by continent. Further, SWGA successfully amplifies genetic regions of interest such as orthologs of P. falciparum drug resistance-associated loci (Pfdhfr, Pfdhps, Pfcrt, Pfk13 and Pfmdr1), and several non-synonymous SNPs were detected in these genes. In conclusion, we have established a robust SWGA approach that can assist whole genome sequencing of P. malariae, and thereby facilitate the implementation of much-needed large-scale multi-population genomic studies of this neglected malaria parasite. As demonstrated in other Plasmodia, such genetic diversity studies can provide insights into the biology underlying the disease and inform malaria surveillance and control measures.

Dataset Information

Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples.

Publications

Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples.

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