Format

Send to

Choose Destination
MBio. 2017 Feb 7;8(1). pii: e02257-16. doi: 10.1128/mBio.02257-16.

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

Author information

1
Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, California, USA.
2
Departments of Medicine and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
3
Universidade Federal de Minas Gerais, Pampulha, Belo Horizonte, MG, Brazil.
4
U.S. Naval Medical Research Unit No. 6, Bellavista, Callao, Peru.
5
Center for Computational Biology and Bioinformatics, UC San Diego, La Jolla, California, USA.
6
Universidad Peruana Cayetano Heredia, Lima, Peru.
7
Public Health England Malaria Reference Laboratory, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
8
Division of Host-Microbe Systems & Therapeutics, Department of Pediatrics, UC San Diego, La Jolla, California, USA ewinzeler@ucsd.edu.

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.

PMID:
28174312
PMCID:
PMC5296604
DOI:
10.1128/mBio.02257-16
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center