Nanopore direct sequencing of mRNA in Trypanosoma brucei Lister427

Trypanosoma brucei causes African sleeping sickness, a fatal human disease. It was hypothesized that long noncoding RNAs (lncRNAs) could be involved in Trypanosoma differentiation from replicative slender form into quiescent stumpy form, thus promoting host survival and parasite transmission. With combined strand-specific and paired-end RNAseq and in silico analysis, 1,428 previously uncharacterized lncRNA candidate transcripts were identified. In order to further investigate the presence, prevalence and potential temporal changes of repertoire of lncRNAs in bloodstream and procyclic form of Trypanosoma, the direct RNA sequencing with Oxford Nanopore device was performed. Using direct RNA sequencing, we confirmed the full length sequence (from splice leader to poly(A) tail) of 1,016 putative lncRNA candidates. The full-length transcripts were filtered out based on the presence of splice leader - nuclear transcripts of trypanosomes undergo trans-splicing which results in addition of short (35-nucleotide) identical nucleotide sequence to their 5' termini. Nanopore sequencing has also revealed, that poly(A) tails of identified lncRNA tend to be longer (most frequently ~112 nt) than the poly(A) tails of the other transcripts (estimated for ~95 nt).