Direct Nanopore Sequencing of two Leishmania infantum JPCM5 life stages

Nanopore Direct RNA Sequencing was performed on FLO-PRO002 (R9.4.1) flow cells using a high-output PromethION 24 platform (ONT, Oxford, United Kingdom). With long Nanopore reads, full-length transcripts can be sequenced end-to-end, facilitating their accurate, unambiguous analysis. Sequencing of polyA RNA from two independent L. infantum promastigote (P1 and P2) and axenic amastigote samples (A1 and A2) was performed. Raw FAST5 were basecalled using Guppy (v6.5.7). The FASTQ were trimmed and aligned to the L. infantum JPCM5 reference genome (v62) using minimap2 (v2.24).We combined Nanopore direct RNA sequencing (DRS) technology to Illumina RNA-Seq (Bioproject PRJNA1071080) to improve existing expression datasets and to gain novel insights into the L. infantum promastigote and amastigote transcriptome diversity and complexity. We exploited the signal sequences for trans-splicing and polyadenylation to accurately delineate the boundaries of L. infantum transcripts. Primary SL and poly(A) sites were mapped in 4487 and 4,566 transcripts, respectively. Furthermore, DRS uncovered multiple processing events occurring mostly within long 3'UTRs, leading to the formation of long non-coding RNAs (lncRNAs). We predicted 1,825 lncRNAs harboring the SL sequence at their 5-end and the polyA tail at their 3-end, of which only 2.1 percent were already annotated in the L. infantum transcriptome. Comparative analysis between the L. infantum promastigote and amastigote transcriptomes depicted 269 developmentally regulated lncRNAs. Interestingly, protein prediction tools combined to mass-spectrometry data identified 64 lncRNAs potentially encoding new proteins. Together, these findings substantially expand knowledge on existing Leishmania expression datasets and provide new important advances on the dynamics of parasite transcriptome diversity and complexity throughout development.