A Robust PacBio Sequencing Technology Based Pipeline in P. cynomolgi Genome Assembly

Background: The first Plasmodium genome sequence was published initially in 2002, for P. falciparum. Genome sequences for other Plasmodium species have followed. Plasmodium coatneyi, which infects Macaca mulatta (rhesus macaques) and serves as a model of P. falciparum, was sequenced recently using PacBio sequencing based assembly (Chien et al. 2016). Building upon on our experience applying PacBio sequencing for this genome assembly, we applied our pipeline to two different strains of P. cynomolgi, B strain and Ceylon strain. Plasmodium cynomolgi serves as a P. vivax malaria model, and like other Plasmodium, its genome has numerous complex multi-gene families and repetitive sequences, which present major difficulties that have prohibited nontelomeric genome assembly with closure with short-read technologies applications. Assembly gaps increase the difficulty of conducting reliable genetics and reference-based gene expression analyses, which are critical for understanding malaria biology and disease progression. We have implemented in house, a PacBio (RSSMRT) technology based pipeline to tackle these issues. Materials and Methods:Genomic DNA from two different strains of P. cynomolgi were extracted from ex vivo matured schizont-stage parasites and SMRTbell DNA libraries (Pacific Biosciences) were constructed according to the PacBio standard protocol with the BluePippin size-selection system (Sage Science). Sequence was generated on a PacBio RSII instrument using P6-C4 chemistry. Following library preparation, HGAP3 de novo assembly was performed on our in-house cluster machine. Contig identity and synteny were evaluated via BLASTn and progressive MAUVE algorithms using the P. knowlesi and P. vivax genome from PlasmoDB as the reference. De novo gene prediction was performed using SNAP and Augustus for gene calls in the MAKER2 genome annotation tool. The annotation was validated with Uniprot, KEGG and OrthoMCL Orthology, and InterProScan5. Results and Conclusions: Our novel PacBio based assembly has been shown to improvePlasmodium genome assemblies, especially in solving gaps or other areas with repetitive regions. In this study, we demonstrated the robustness of our pipeline on two strains of P. cynomolgi, which are important for studies modeling P. vivax. This technology out-performs short-read technology, and the new assemblies present more insights into subtelomeric regions. This technology has also been applied to a strain of P. vivax that is relevant for our team’s study of hypnozoites and relapse biology.