NEW YORK (GenomeWeb) – A team led by researchers at the University of California, San Diego and the University of Pennsylvania has demonstrated that a targeted amplification technique can be used to sequence the genomes of malaria parasites found at low levels in human blood samples.
With the approach, called selective whole-genome amplification (SWGA), "you don't need any processing at the time of sample collection, so you can send the blood samples and extract the DNA off-site later," co-first author Annie Cowell, an infectious disease research fellow in the lab of senior author Elizabeth Winzeler at UCSD, said in an interview.
More broadly, the method should be adaptable to other infectious disease agents, since finding ways to nab parasite DNA out of a complicated mixture overrun with host DNA is a challenge, especially for parasites that do not cooperate with being cultured in the lab.
"Wherever you have a parasite infecting a host and you want to sequence the parasite, this is a good option for that," said Jane Carlton, a researcher at New York University and a malaria genomics expert who was not involved in the study.
For their new study, published in mBio this week, Cowell, Winzeler, and colleagues tweaked SWGA to preferentially amplify DNA from the tricky-to-culture human malaria parasite Plasmodium vivax — an approach they successfully used to sequence the genomes of P. vivax isolates in nearly two dozen unprocessed blood samples or blood spots from infected patients in Peru.
As the name implies, the SWGA method preferentially amplifies pathogen DNA over host DNA without physically dividing parasite and host cells. It does so by using short primers that target sequence motifs that are abundant in the pathogen genome, but rare or absent in the host genome — in this case, the human genome.
Those primers are then paired with a DNA polymerase called phi29 that has a strand displacement function, generating long amplification products, Cowell explained.
The general approach and primer design strategy used for the P. vivax analysis built on an SWGA method that researchers from UPenn described in 2014 for amplifying and sequencing DNA from a Wolbachia bacterial symbiont found inside fruit flies.
UPenn's Beatrice Hahn, a coauthor on the new mBio paper, led a study reported in Nature Communications last year that adapted SWGA to P. falciparum, a particularly deadly malaria parasite in humans, and to other Plasmodium species. The approach worked well enough to sequence Plasmodium DNA from asymptomatic chimpanzees with low levels of the parasites in their blood.
Authors of the new mBio study "have taken the next logical step, which is to design primers for P. vivax, too," said Carlton in an interview. "It's another piece in the armory that we have, to try to work with this parasite that is present in such small quantities in blood samples."
The method is expected to be particularly useful in situations where there is not sufficient time, staff, or infrastructure to enrich for parasite DNA with alternative means. Although white blood cell filtering, or leukocyte depletion, is an inexpensive strategy to weed out uninfected host blood cells and enrich for malaria parasites, for example, it requires fresh blood samples and is not always an option in the field. On the other hand, molecular enrichment methods such as RNA-bait-based capture are often pricier and may not be suitable to resource-limited locales.
"All of the different techniques for parasite enrichment or human DNA depletion have their advantages and disadvantages," Carlton said. "It really depends what the lab wants to do and where they're located in the world."
Cowell noted that several researchers have already been in touch about the P. vivax SWGA method, keen to take a crack at using the SWGA method on their own malaria samples.
"I think a lot of people get samples that haven't been leukocyte-filtered or are leukocyte-filtered and have human contamination," she noted. "People are really excited to try to amplify the P. vivax DNA using this in order to get a better look at those parasites and get more information."
The P. vivax genome was described in a pair of studies by independent teams in Nature in 2008, more than half a decade after investigators from the Institute for Genomic Research, the Wellcome Trust Sanger Institute, and elsewhere published an analysis of the P. falciparum genome.
For a population study of P. vivax that was published in Nature Genetics in 2012, NYU's Carlton and her colleagues tackled P. vivax isolates from northwest Africa, South America, South Asia, and East Asia that were adapted to growth in monkeys, where it's possible to get higher P. vivax parasitemia. That analysis revealed particularly high genetic diversity in P. vivax.
An international research team reporting in PLOS Neglected Tropical Diseases later that year turned to leukocyte depletion and deep sequencing to generate P. vivax sequences directly from samples of malaria patients in Madagascar or Cambodia, while researchers reporting in Genome Research in 2014 used a single-cell sequencing strategy to enrich for malaria parasite DNA and explore its heterogeneity.
More recently, Carlton led a team that used sticky bait-based capture for grabbing P. vivax DNA out of patient samples. That approach was applied to 182 P. vivax isolates from around the world for a paper published in Nature Genetics last year.
Her NYU team is investigating amplification-free genome sequencing approaches for P. vivax that rely on the Oxford Nanopore MinION sequencer. The group is exploring methylation-based options for enzymatically chopping up and removing human DNA, while capturing DNA from malaria parasites that tends to be less methylated.
In an effort to add to malaria parasite enrichment resources available or under development, the authors of the mBio paper carefully designed and tested a set of primers for applying SWGA to P. vivax.
Despite some similarities between P. falciparum and P. vivax, adapting the amplification method to P. vivax was no trivial feat, Cowell explained, in part due to differences in the GC-content in the malaria genomes.
"P. vivax is much more GC-rich, with about 45 percent GC-content, compared to P. falciparum, which is 19 percent," she said. "With P. vivax, we had to actually design GC-enriched primers that were targeted more towards GC-rich parts of the genome to get better amplification of the DNA."
The team initially narrowed in on a set of 222 primers. After weeding out primers with inappropriate melting temperatures, homodimerization features, or human genome binding proclivities and testing sets of half a dozen primers at a time, the group selected 10 primers for P. vivax SWGA followed by genome sequencing.
That primer set resulted in lower-than-anticipated coverage over GC-rich parts of the genome, prompting a primer redesign to better target such regions, informed by sequences from the P. vivax reference genome.
When they used that primer set to amplify and sequence P. vivax isolates in 18 unprocessed blood samples and four dried blood spot samples from malaria patients in Peru, the researchers generated sequences covering the P. vivax genome to nearly 24-fold, on average, in the blood samples.
Genomes from the blood spots had average depths of 16-fold. About half of the P. vivax genome could be called in the samples, on average, and the team reported that the SWGA-assisted genome sequences compared favorably to those generated for 10 white blood cell-filtered blood samples.
"Using SWGA, we achieved a higher-than-average callable P. vivax genome than was obtained for leukocyte-depleted clinical samples sequenced at similar depth," the authors wrote.
Because some parts of the P. vivax genome are amplified more readily than others, genome sequence data generated after SWGA is not suited for analyzing copy number variants, Cowell noted. But the researchers were able to identify SNPs and small insertions and deletions in the P. vivax genomes — information that can be used for everything from relatedness between parasites to drug resistance patterns.
Cowell noted that she is now attempting to apply the approach for comparing P. vivax in the same person over time to see how the parasite population changes, if at all.
She and her co-authors noted that applying whole-genome sequencing more routinely in the clinic will help in distinguishing new malaria infections from cases that involve re-infection or re-emergence of malaria-causing parasites that had been dormant in the livers of symptom-free human hosts.