Selective Whole Genome Amplification Shows Potential for Sequencing Avian Malaria Pathogen Genomes

• Ishika Sharma, Applied Molecular Biology & Biotechnology, University of Delaware

• Vincenzo Ellis, Entomology and Wildlife Ecology, University of Delaware

Understanding the genetic makeup of pathogens is crucial for disease diagnosis, vaccine development, outbreak prediction, and implementing effective preventative measures. However, sequencing pathogen genomes in the presence of host DNA presents a significant challenge due to the abundance of host DNA compared to the relatively small amount of pathogen DNA. Traditional techniques like polymerase chain reaction (PCR) target particular genes and can therefore miss important regions of the pathogen’s genome, hampering comprehensive analysis. In this study, we present a selective whole genome amplification approach to sequence the complete genome of the avian malaria pathogen Plasmodium relictum, specifically genetic lineages SGS1 and GRW11. To overcome the abundance of host DNA in avian blood samples, we employed the Phi29 enzyme with primers specifically designed to bind more readily to the pathogen’s genome than to the host’s. Using an isothermal amplification process, we successfully prepared the amplified pathogen genomes for Illumina sequencing on an Illumina MiSeq platform. Additionally, we applied this protocol to DNA extracted from the blood of a wood thrush (Hylocichla mustelina) infected with the pathogen P. unalis (lineage TUMIG03) and a negative water control. By mapping the resulting sequences to the P. relictum reference genome, we found that we could use this technique to sequence the full genomes of SGS1 and GRW11, although the latter exhibited patchy sequence coverage. While TUMIG03 showed limited amplification, some sequences were identified as Plasmodium but could not be mapped to the reference due to the use of the likely very different P. relictum reference genome. Our results demonstrate that selective whole genome amplification holds promise as an approach to investigate avian malaria pathogen genomics, thus shedding light on their evolution. These findings have significant implications for setting up preventative measures against pathogenic diseases. By understanding the genomic characteristics and evolutionary patterns of pathogens, we can design more effective preventative strategies, contributing to the advancement of public health.