​cgi?​taxid=​5833and PlasmoDB [23] databases. The remaining 14 insertions either mapped to telomeric repetitive elements or could not be mapped to a chromosomal location through BLAST SB273005 solubility dmso searches of public databases. The identifiedpiggyBacinsertion sites were distributed throughout LOXO-101 ic50 the

genome in all 14P. falciparumchromosomes (Fig.2a) with no bias for any particular chromosome (Fig.2b). AllpiggyBacinsertions were obtained in the expected TTAA target sequences except two that integrated into TTAT and TTAG sequences. As in other organisms [17,20],piggyBacpreferentially inserted into predicted transcribed units ofP. falciparumgenome (Fig.3a), affecting 178 transcription units. Thirty-six of the insertions resulted in direct disruption of open reading frames (ORFs) and 3 insertions 4SC-202 purchase were mapped to introns. A vast majority of insertions (119) occurred in 5′ untranslated regions (UTRs) whereas only a few (22) were obtained in 3′ UTRs (Additional file 1). Figure 2 Distribution of piggyBac insertion

sites in the P. falciparum genome.(a)A representation of the 14P. falciparumchromosomes withpiggyBacinsertion loci (represented by red vertical lines) shows extensive distribution ofpiggyBacinsertions through out the parasite genome.(b)Comparison of chromosomal distribution ofpiggyBacinsertions to the percent genome content of each chromosome shows unbiased insertions intoP. falciparumgenome. Plot and curve fits of percentpiggyBacinsertions and percent chromosome size are depicted in the inset. Figure 3 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 oxyclozanide inPlasmodium[48,49]. (b) Comparison of gene functions of all annotated genes in the genome (outer circle) to genes inpiggyBac-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 inpiggyBac-inserted

loci (inner circle) validates the ability ofpiggyBacto insert in genes expressed in all parasite life cycle stages. (d) A comparison ofpiggyBac-inserted TTAA sequences to TTAA sequences randomly selected from the genome showed preferential insertion ofpiggyBacinto 5′ UTRs of genes (asterisk- χ2test, df 1, P = 1.5 × 10-12) whereas a significantly lower number of insertions were observed in CDS and introns (double asterisks- χ2test, df 1, P = 1.09 × 10-13). piggyBacinserts randomly into all categories of genes with a strong preference for 5′ untranslated regions Obtaining unbiased insertions into the genome is critical for whole-genome mutagenesis and other large-scale analyses. Hence, we evaluated the randomness ofpiggyBacinsertions into theP.