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.

Population genomic evidence of a Southeast Asian origin ofPlasmodium vivax

“…To overcome host DNA contamination, selective whole genome amplification (sWGA) was used to enrich submicroscopic DNA levels as already described elsewhere ( 53 ). This technic preferentially amplifies P. vivax and P. vivax-like genomes from a set of target DNAs.…”

“…This technic preferentially amplifies P. vivax and P. vivax-like genomes from a set of target DNAs. For each sample, the DNA amplification was carried out by the strand-displacing phi29 DNA polymerase and two sets of P. vivax -specific primers that target short (6 to 12 nucleotides) motifs commonly found in the P. vivax genome (set1920 and PvSet1) ( 52, 53 ). For each set of primers separately, 30 ng of input DNA was added to a 50 µl reaction mixture containing 3.5 µM of each sWGA primers, 30 units of phu29 DNA polymerase enzyme (New England Biolabs), 1X phi29 buffer (New England Biolabs), 4 mM of dNTPs (Invitrogen), 1% of Bovine Serum Albumine and sterile water.…”

Malaria Epidemiology at the Clone Level

“…Sequencing from blood samples is hampered by an >99% excess of human DNA. It is thus restricted to high-density infections with leucocyte depletion at the point of collection [94], or alternatively enrichment of parasite DNA over human DNA after extraction is required [95, 96]. …”

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

“…Some studies separate target sequences from total DNA or RNA by exploiting base modifications or transcriptional properties specific to the pathogen 15 , vector 16 or host 17,18 . Others describe the use of biotinylated hybridization probes 19–22 or selective whole-genome amplification, e.g., based on the strand displacement function of phi29 DNA polymerase 23 . Such techniques are costly and often excessive when a study’s primary objective is to evaluate genetic distances and diversity among samples rather than to reconstruct complete haplotypes or investigate structural genetic traits.…”