Abstract
High throughput sequencing techniques are poorly adapted for in vivo studies of parasites, which require prior in vitro culturing and purification. Trypanosomatids, a group of kinetoplastid protozoans, possess a distinctive feature in their transcriptional mechanism whereby a specific Spliced Leader (SL) sequence is added to the 5′end of each mRNA by trans-splicing. This allows to discriminate Trypansomatid RNA from mammalian RNA and forms the basis of our new multiplexed protocol for high-throughput, selective RNA-sequencing called SL-seq. We provided a proof-of-concept of SL-seq in Leishmania donovani, the main causative agent of visceral leishmaniasis in humans, and successfully applied the method to sequence Leishmania mRNA directly from infected macrophages and from highly diluted mixes with human RNA. mRNA profiles obtained with SL-seq corresponded largely to those obtained from conventional poly-A tail purification methods, indicating both enumerate the same mRNA pool. However, SL-seq offers additional advantages, including lower sequencing depth requirements, fast and simple library prep and high resolution splice site detection. SL-seq is therefore ideal for fast and massive parallel sequencing of parasite transcriptomes directly from host tissues. Since SLs are also present in Nematodes, Cnidaria and primitive chordates, this method could also have high potential for transcriptomics studies in other organisms.
Highlights
Trypanosomatidae are a family of protozoan organisms that comprise a variety of human and animal pathogenic species including human/animal African trypanosomiasis or sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi) and surra (Trypanosoma evansi)
In this study 16% more differentially expressed genes (DEGs) were observed for the Illumina stranded mRNA seq than spliced leader (SL)-seq, this can be fully explained by the fact that the Illumina libraries were sequenced deeper
We presented and validated a mRNA profiling method called SL-seq that we developed to sequence Trypanosomatid transcriptomes directly from the infected host cells in a high-resolution and high-throughput fashion
Summary
Trypanosomatidae are a family of protozoan organisms that comprise a variety of human and animal pathogenic species including human/animal African trypanosomiasis or sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi) and surra (Trypanosoma evansi). Sequencing data obtained with SL Trapping slightly differs from conventional RNA-seq data, since sequencing reads map often just upstream of the gene (5′ UTR), where the spliced leader is added. This requires a bioinformatics pipeline that is adapted and benchmarked for this type of data. This work could bring transcriptomics in parasitic Trypanosomatids to a new performance level, opening a novel avenue for in-depth understanding of their molecular regulation This method has high potential for in vivo studies of other symbionts with a SL-containing mRNA such as nematodes, trematodes, dinoflagellates and primitive chordates[13]
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