Fig 1.
Growth history of cells used for RNA extraction.
Data shows growth curve for one of the three replicates. L. mexicana promastigotes (PRO) were maintained in exponential growth by diluting the culture to 1x106 cells ml-1 every day (blue line). A second promastigote culture was inoculated with 1–2.5x105 cells ml-1 and left to grow for five days to stationary phase (red line). Stationary phase promastigotes were used to infect bone marrow derived macrophages (BMDM) to produce intracellular amastigotes (AMA) or differentiated to axenic amastigotes (AXA) in Schneider’s medium (SM5.5) for 24h. RNA was extracted from AMA, AXA and PRO on day 7.
Table 1.
Estimate of L. mexicana RNA in mixed RNA samples from infected BMDMs.
Table 2.
RNA-seq read mapping summary.
Fig 2.
Distribution of SLAS, PAS and UTR lengths.
(A) Distribution of assigned SLAS numbers per gene. A SLAS was assigned if at least nine SL-containing reads were mapped to this position across all nine random primed libraries (n = 20,812). (B) Distribution of assigned PAS numbers per gene (n = 95,097). A PAS was assigned if at least six reads terminating in ≥ 5 A were mapped to this position across all six T15VN primed libraries. (C) Distribution of 5’ UTR lengths (without the SL sequence; n = 9,029). (D) Distribution of 3’ UTR lengths (n = 9,029).
Fig 3.
Characterisation of novel transcripts.
(A) Size distribution of novel transcripts (n = 936). (B) Size distribution of transcripts derived from genes annotated in TriTrypDB V6 (n = 8,250). (C) Size distribution of largest ORFs found on the sense strand of novel transcripts (n = 936). (D) Size distribution of CDS annotated in TriTrypDB V6 (n = 8,250).
Fig 4.
Conservation of novel transcript sequences.
(A) The novel transcripts were used as query sequences in a reciprocal best tblastx search of 12 kinetoplastid genomes. “RBB hits total” indicates the number of reciprocal best tblastx hits returned; “log10 E ≤ -20” indicates the number of hits returned with an E value ≤ 10−20 for the reciprocal tblastx search. (B) Venn diagram showing the number of hits returned in a series of reciprocal best tblastx searches comparing the novel transcripts found in L. mexicana (this study), L. major [30] and T. brucei [42].
Fig 5.
Conservation of novel transcript sequences across kinetoplastid genomes.
The 936 novel L. mexicana transcripts and 7 control genes were used as queries in tblastx searches of 12 kinetoplastid genomes and the best hits were then used in a reciprocal tblastx search against the complete L. mexicana genome. The heat maps indicate the E value of the returning hits, with darker shades of blue representing lower E values. Sequences that did not return a hit are represented in red. (A) Sequences used as positive controls for conserved CDS (Gene IDs: PFR2, LmxM.16.1430; gGAPDH, LmxM.29.2980; γ-tubulin, LmxM.25.0960; SAS-6, LmxM.34.4280; RPB12, LmxM.20.0490; SmD2, LmxM.32.3190; APX, LmxM.33.0070). (B) Intergenic sequences downstream of the CDS in (A), used as negative controls. (C) Each row represents one of the 936 novel L. mexicana transcripts.
Fig 6.
The majority of highly expressed transcripts are shared between AMA, AXA and PRO.
The Venn diagram shows for each cell type the 91 transcripts comprising the top percentile of FPKM values and indicates the extent of overlap between the three datasets.
Fig 7.
Differential gene expression between AMA, AXA and PRO.
(A) Table summarising the number of differentially expressed (DE) genes in each pair-wise comparison; “novel” refers to the 936 novel transcripts defined in this study. (B-D) Volcano plots for the comparisons between PRO and AMA (B), PRO and AXA (C), AXA and AMA (D). Each dots represents one transcript; red denotes differential expression (padj ≤ 0.05). Arrows indicate the cell type showing higher expression.
Fig 8.
Distribution of differentially expressed genes across chromosomes.
Maps of the 34 L. mexicana chromosomes show the location of genes that are preferentially expressed in AMA (red), PRO (blue) or constitutively expressed (grey).