Genomic sequence capture of Plasmodium relictum used to describe population structure of related lineages. Genomic sequence capture of Plasmodium relictum in experimentally infected birds

Sequencing parasite genomes in the presence of host DNA is challenging. Sequence capture can overcome this problem by using RNA probes that hybridize with the parasite DNA and then are removed from solution, thus isolating the parasite DNA for efficient sequencing. Here we describe a set of sequence capture probes designed to target 1036 genes (ca. 2.5 Mbp) of the globally distributed avian haemosporidian parasite, Plasmodium relictum. Previous sequence capture studies of avian haemosporidians from the genus Haemoproteus have shown that sequencing success depends on parasitemia, with low intensity, chronic infections (typical of most infected birds in the wild) often being difficult to sequence. We evaluate the relationship between parasitemia and sequencing success using birds experimentally infected with P. relictum and kept under laboratory conditions. We confirm the dependency of sequencing success on parasitemia; the bird with the lowest parasitemia (0.1% infected red blood cells) had only 3.36% of targeted nucleotides sequenced to a depth of coverage of 5X. Sequencing success increased nonlinearly such that in a bird with parasitemia of 0.24%, 26.39% of targeted nucleotides were sequenced to a depth of coverage of 5X. Plasmodium relictum is composed of multiple lineages defined by their mitochondrial DNA haplotype including three that are widespread (SGS1, GRW11, and GRW4); the probes described here successfully isolated DNA from all three. Furthermore, we used data from 25 genes to describe both among and within lineage genetic variation. For example, two samples of SGS1 isolated from different host species differed by 9 substitutions across those 25 genes. The sequence capture approach we describe will allow for the generation of genomic data that will contribute to our understanding of the population genetic structure and evolutionary history of P. relictum, a widespread and extreme host generalist parasite species.