5CAPture-seq: a method for enriching full-length cDNA and identifying 5′ capped nucleotides

Current transcriptomic methods for mapping 5′-ends of eukaryotic RNA employ only 5′-end enriched samples. The absence of a matched unenriched control limits the ability to distinguish genuine 5′ ends from incomplete cDNAs. We developed a transcriptomic method based on sequencing of cDNA enriched for full-length fragments and an unenriched control. From the mapped sequence reads, enrichment scores are calculated for all transcribed nucleotides and a statistical model of the enrichment constructed. We tested the method in the human malaria parasite Plasmodium falciparum. Data were obtained from ring, trophozoite and schizont stages of the parasite intra-erythrocytic development cycle. Two groups of 5′ capped nucleotides were assigned by unsupervised clustering. The first group contains sites located mostly outside of annotated protein-coding exons in regions of high local nucleosomal occupancy, but low occupancies of ribosome and elongating RNA polymerase II. The majority of sites in the second group are intra-exonic and show different patterns, most notably a peak of ribosome occupancy centered on the 5′ end position and a biased representation among codons possibly prone to ribosome stalling. Our method can be used to annotate 5′ capped nucleotides, including intra-exonic nucleotides that can be distinguished from incomplete cDNA artifacts. Total RNA was extracted from synchrononous cultures of Plasmodium falciparum. Parasites were harvested at ring, trophozoite and schizont stages. Two independent cultures were performed. cDNA from each RNA sample was synthesized and split into two pools. One pool was enriched for full-length cDNA, whereas another was processed as an unenriched control sample. Enriched and unenriched cDNAs were converted to Illumina sequencing libraries for transcriptomic sequencing.