Identification and characterization of five types of NATs in Tetrahymena. (A) Schematics for NATs annotation. NATs were identified on the TGD2024 using both transcriptomic and epigenetic data. Identified NATs were further categorized based on their relative positions to corresponding sense transcripts. (B–E) IGV snapshots showing five types of NATs. They included promoter NATs (B), originating from shared bidirectional promoters of the sense transcripts; type 1 exonic NATs (C), located within 1 kb downstream of the TSSs of the sense transcripts and sharing epigenetic marks with their sense transcripts; type 2 exonic NATs (D), located > 1 kb downstream of the TSSs of the sense transcripts; and intronic NATs (E), transcribed from the intronic regions of sense transcripts. (F) Distribution profiles of H3K4me3, H2A.Z, 6mA and well-positioned nucleosomes on the transcript body of NATs. Transcripts were scaled to unit length and were extended to each side by 1 kb length. (G) Box plot representation of normalized read counts for sense transcripts and NATs during growth, starvation and conjugation. (H) An IGV snapshot showing the anti-correlation of temporal expression patterns between a NAT and its corresponding sense transcript (left). The line chart (right) depicted the proportion of expression level for sense and antisense transcripts at different time points. The error bar represented the standard deviation. (I) The box plot showing that the ASD of NATs exceeded that of their sense transcripts (the median of NATs and sense transcripts were 0.96 and 0.28, respectively). Mann–Whitney was performed. ****P< 0.0001. ASD was defined as the number of different types of splice sites divided by the total reads aligned to the NATs or sense transcripts.