piggyBac insertions in the genome are random but preferentially occur in 5' untranslated regions. (a) Genomic transcription units were defined to include 2 kb of 5' UTR, the coding sequence, the introns and 0.5 kb of 3' UTR, based on previous studies in Plasmodium [48, 49]. (b) Comparison of gene functions of all annotated genes in the genome (outer circle) to genes in piggyBac-inserted loci (inner circle) shows an equivalent distribution confirming random insertions in the parasite genome. (c) Comparison of stage-specific expression of all annotated genes (outer circle) to those in piggyBac-inserted loci (inner circle) validates the ability of piggyBac to insert in genes expressed in all parasite life cycle stages. (d) A comparison of piggyBac-inserted TTAA sequences to TTAA sequences randomly selected from the genome showed preferential insertion of piggyBac into 5' UTRs of genes (asterisk- χ2 test, df 1, P = 1.5 × 10-12) whereas a significantly lower number of insertions were observed in CDS and introns (double asterisks- χ2 test, df 1, P = 1.09 × 10-13).