The 1% solution: Decoding malaria genomes

Imagine the sample you want to study consists of dried blood on filter paper stored for more than a year at room temperature in Senegal. Keep in mind that what you’re looking for – DNA of the one-celled parasite that causes malaria – typically amounts to 1 percent of the genetic material you have in hand, outnumbered by human DNA that makes up the other 99 percent of that blood spot.

Sequencing samples never intended for DNA analysis is daunting, but even blood drawn from the arm of an infected patient and promptly shelved in a research-quality freezer can be difficult to use, for the same reason: Separating human cells from the invader cells you care about is still a barrier. In the case of malaria, the need is urgent. Infections by one malaria parasite, Plasmodium falciparum, kill more than 800,000 people a year in sub-Saharan Africa, most of them children under 5 years old.

Scientists at the Broad have met the challenge of parsing this parasite’s genome by adapting “hybrid selection,” a protocol designed for another nearly 99-to-1 mismatch: the human genome and its tiny fraction of protein-encoding genes, called the exome. Writing in Genome Biology, senior author Dan Neafsey and his colleagues report on the technique in this paper, suggesting it has potential beyond malaria to rapidly glean DNA data during an infectious disease outbreak or analyze old samples to help solve the riddle of drug resistance.

“Just as you can do genome-wide association studies in humans to find the genes that underlie common diseases or other traits, you can do the same thing in the genomes of malaria parasites to find the genes that confer resistance to drugs, or virulence, or other characteristics that affect public health,” said Dan, group leader of malaria genome sequencing and analysis in the Broad’s Gene Sequencing and Analysis Program. “I was curious to see whether we could use hybrid selection in a slightly different application to efficiently target for sequencing just pathogen DNA that one finds in clinical samples from infected patients.”

Hybrid selection, a far-reaching advance devised by Broad scientists led by Andreas Gnirke and Alexandre Melnikov and reported in 2009, is a way to fish for the specific DNA molecules swimming in the larger DNA pond by using baits complementary to the targets in the exome they want to sequence. After reeling in their desired DNA, which “stick” to matched base-pair baits, they wash away the by-catch they don’t need. The protocol allows less costly and more efficient exome sequencing than whole genome sequencing, making hybrid selection a tool now widely used in the field.

When Dan and his team tried hybrid selection on clinical malaria samples, they created baits two ways. One approach was to use commercially available stretches of DNA to enrich what was found in their samples. But then Alex and Peter Rogov suggested using pure DNA from the entire malaria genome that had previously been cultured.

“If you use a solution of whole genomic DNA to start with, you automatically get everything and you don’t have to make separate orders of batches to cover the entire genome,” Dan said. “This lets you very efficiently come up with a bait set that covers the entire pathogen genome that you want to recover.”

When the scientists tried this approach, the amount of malaria DNA in a sample was typically enriched 40-fold. That means a sample that started out as 1 percent malaria ended up being close to half of the total DNA.

The sample from Senegal on which they tested the technique was more difficult. Malaria DNA made up only 0.1 percent of the dried blood spot. But using hybrid selection, they were able to boost the level of malaria DNA in the sample to 6 percent. When they did a second round of hybrid selection on that, the malaria DNA fraction jumped to 47.5 percent.

“That wasn’t too shabby,” Dan said. “This was a very good way of rescuing samples that otherwise would not be considered fit for sampling.”

Dan’s group will turn next to a form of malaria called Plasmodium vivax, which does not cause as many deaths as P. falciparum but brings misery around the world to those who contract it. P. vivax is almost impossible to culture, so hybrid selection may play a huge role in sequencing the genomes of its many strains.

“It’s a particularly ripe application, I think, for this hybrid selection protocol,” Dan said.