Analysis of mRNA processing at whole transcriptome level, transcriptomic profile and genome sequence refinement of Trypanosoma cruzi

Abstract

The genomic sequence of Trypanosoma cruzi, the protozoan causative of Chagas disease was published more than a decade ago. However, due to their complexity, its complete haploid predicted sequence and therefore its genetic repertoire remains unconfirmed. In this work, we have used RNAseq data to improve the previous genome assembly of Sylvio X10 strain and to define the complete transcriptome at trypomastigote stage (mammalian stage). A total of 22,977 transcripts were identified, of which more than half could be considered novel as they did not match previously annotated genes. Moreover, for the first time in T. cruzi, we are providing their relative abundance levels. We have identified that Sylvio X10 trypomastigotes exhibit a predominance of surface protein genes, specifically those encoding trans-sialidase and mucin-like proteins. On the other hand, detailed analysis of the pre-mRNA processing sites revealed some similarities but also some differences in the spliced leader and different polyadenylation addition sites compared to close related kinetoplastid parasites. Our results also confirm that transcription is bidirectional as occur in other kinetoplastids and the proportion of forward-sense and reverse-sense transcripts is almost equivalent, demonstrating that a strand-specificity does not exist.

Figure 1

Genome improvement summary. Read depth mapping, coverage, and changes applied to the previous genomic sequence of Sylvio X10.

Figure 2

Transcriptomic assembly and annotation. Figure shows 31 kb spanning region of chromosome 2. (A) Sequencing coverage and reads aligned to reference genome, (B) Sequence Leader-containing reads mapped to reference, (C) Genomic and transcriptomics annotations: primary annotations corresponds to genomic annotations from companion and Stringtie, primary assembly (Stringtie) and final transcriptomic annotations.

Figure 3

Coding sequence of Sylvio X10 C-LS. Previously defined (by genomic annotation) and the coding sequence defined by RNAseq data.

Figure 4

Gene ontology classification (Level three) of the Sylvio X10 transcriptome. Families containing at least 10 mapped transcripts were considered.

Figure 5

Genomic composition (by nucleotide frequencies) on SL and polyadenylation insertion sites. (A) SL addition sites (n = 18,589). (B) PolyA-tail addition sites (n = 9,789).