Fig. 1

Overview of P. falciparum RNA sequencing sample set, computational pipeline, and read alignment metrics. (A) We harvested total RNA from two independent P. falciparum blood stage time courses, including a 56-hour time course consisting of eleven samples. We combined samples harvested 4 and 8 hpi at equal ratios (further referred to as T6). Similarly, we combined samples harvested 12 and 16 hpi at equal ratios (further referred to as T14). We harvested four additional samples from a second time course approximately 4 h before and after gross stage transitions. Thus these samples correspond to the late ring, early trophozoite, late trophozoite, and early schizont stages, respectively. In total, we sequenced fifteen strand-specific RNA sequencing (RNA-seq) libraries on an Illumina Hiseq 2000 machine. Illumina sequencing yielded approximately 614 million 101-bp paired-end reads. We analyzed reads using the Tuxedo suite (Bowtie, TopHat, Cufflinks, Cuffmerge, and Cuffdiff) and according to the circBase circRNA discovery pipeline [85]. Using this approach, we identified 660 intergenic lncRNA (647 unique loci), 474 antisense RNA (467 unique loci), and 1381 circRNA candidates. Additionally, 3815 genes, 127 transcripts, and 81 promoters reached statistical significance in terms of differential expression, alternative splicing, and alternative promoter usage, respectively. (B)/(C) Normalized read alignment tracks across a PfEMP1-encoding var gene [PlasmoDB:Pf3D7_0412700] and the CLAG3.1 gene [PlasmoDB:Pf3D7_0302500] indicated that these challenging loci could generally be (perfectly and uniquely) mapped. Annotated gene models are shown in dark green and dark blue. Reads from each 56-hour time course sample mapping to the (−) strand are shown below each horizontal axis in light green, while reads mapping to the (+) strand are shown above each horizontal axis in light blue. Uniqueness of 100mers is plotted in red as a mappability track, where the baseline represents a score of one, or uniquely mapping. (D)/(E) Plotting the expression during the 56-hour time course of the dominant PfEMP1-encoding var gene [PlasmoDB:Pf3D7_0412700] and both the CLAG3.1 [PlasmoDB:Pf3D7_0302500] and CLAG3.2 [PlasmoDB:Pf3D7_0502200] genes showed, respectively, that var gene expression peaked during the ring stage, whereas CLAG3.1 and CLAG3.2 expression peaked during the schizont stage. Moreover, as CLAG3 genes are mutually exclusively expressed [27, 28], we found that that the bulk of our parasites transcribed only the CLAG3.1 gene. Expression is plotted in units of log2(FPKM + 1). (F) The percent of reads in each library mapping to annotated transcripts in the proper orientation (per reads mapping to annotated transcripts) ranged from 98.92 % to 99.81 %. The average calculated from both reads is reported