The random shotgun approach was used to obtain roughly eightfold coverage of the whole nuclear genome sequence from the erythrocyte stage of the Pk1(A+) clone of the H strain of P. knowlesi​5 (link). Sequence reads were assembled (as described in the Supplementary Information) and positional information from sequenced read pairs were used to resolve the orientation and position of the contigs. The assembled P. knowlesi contigs were iteratively ordered and oriented by alignment to P. vivax assembled sequences (described in ref. 4 ) and by manual checking. Automated predictions from the gene finding algorithms were manually reviewed by comparison to orthologues in other Plasmodium species. Artemis and Artemis Comparison Tool (ACT) were used (as described previously28 (link)) for annotation and curation and viewing the TBLASTX comparisons of regions with conserved synteny between P. knowlesi, P. vivax and P. falciparum. This also allowed us to curate gene models and identify local interruptions of synteny. Functional annotations were based on standard protocols as described previously6 (link).