NEW YORK (GenomeWeb) - An international team led by researchers at the University of Pennsylvania has used selective whole-genome amplification to sequence three whole genomes of chimpanzee parasites — one Plasmodium reichenowi and two Plasmodium gaboni genomes — that are distantly related to Plasmodium falciparum, which causes malaria in humans.
The findings, published in Nature Communications, validated the researchers' selective amplification method for characterizing cryptic pathogen species, and revealed evolutionary events that likely led to P. falciparum colonizing human hosts.
The parasite species that were sequenced all fall into a subgenus called Laverania. There are seven species in this subgenus, six in apes (chimpanzees and gorillas) and one in humans, Beatrice Hahn, a professor of medicine at the University of Pennsylvania and corresponding author on the study, told GenomeWeb. The closest living relative to P. falciparum is one that only resides in gorillas; however, the researchers were not able to access blood samples from this species, since they are protected due to their endangered status.
As such, the researchers decided to focus on the chimpanzee parasites. While chimpanzees are also a protected species, the researchers got permission to use leftover blood samples from annual physicals provided by the Sanaga-Yong Chimpanzee Rescue Center. Since they were working with small amounts of blood and smaller amounts of parasite DNA, they had to use a sequencing method that allowed them to get whole genomes from limited samples which is why they choose to selectively amplify the parasite genomes.
It's a technique originally developed in the lab of Dustin Brisson, a professor in the department of biology at the University of Pennsylvania and co-author on the paper. The technique uses specially designed primers that bind frequently to the Plasmodium genomes and very rarely to other genomes alongside ϕ29, a polymerase capable of strand displacement and has high processivity, said Sesh Sundararaman, a student in the medical scientists training program at the University of Pennsylvania and first author on the study.
Once they created their primers, the researchers selectively amplified P. gaboni and P. reichenowi genomes and sequenced them on Illumina's MiSeq platform. They mapped their reads based on PrCDC and Pf3D7 reference sequences to generate draft genomes for both parasites.
The researchers then performed genome-wide analyses and found that they were able to create three whole genomes, except for sub-telomeres, which the researchers attributed to a problem of assembly, not amplification. Hahn told GenomeWeb that they hope to use Pacific Biosciences technology in future testing, since it provides much longer reads that she believes will rectify the assembly issue.
They found that these genomes did in fact represent distinct species that hadn't been previously verified, and that there was no evidence of cross-species mating. The researchers observed that both parasite species are about 10 times more genetically diverse than P. falciparum, which they noted indicates that the human parasite "has been through a severe genetic bottleneck, consistent with a very recent origin.".
However, what the researchers found particularly notable was the Laverania-specific expansion of a multigene family involved in erythrocyte remodeling, and they showed that a short region on chromosome 4, which encodes two functionally related essential invasion genes, was horizontally transferred into a recent ancestor of P. falciparum.
Importantly, they were able to show that selective whole-genome amplification could be used with more complex organisms than bacteria. Additionally, the researchers stated in the paper that this method could be adapted to other microbes and/or host species when a reference genome is available.
Sundararaman told GenomeWeb that while this could be used to investigate parasites in rare species cases, it has many other potential applications. In a resource-poor setting, it may only be possible to obtain a small amount of blood and when researchers don't have any materials to purify the parasite on hand. However, this method allows researchers to design primers that can accommodate that, he explained.
Sundararaman and his colleagues have made a prototype of their primer design pipeline available to be used for selective whole-genome sequencing in other organisms.