Multiplexed Spliced-Leader sequencing: a high-throughput, selective method for RNA-seq in Trypanosomatids

Bart Cuypers, Malgorzata Domagalska, Pieter Meysman, Géraldine de Muylder, Manu Vanaerschot, Hideo Imamura, Franck Dumetz, Thomas Wolf Verdonckt, Peter J Myler, Gowthaman Ramasamy, Kris Laukens, Jean-Claude Dujardin

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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.

Original languageEnglish
Article number3725
JournalScientific Reports
Volume7
Number of pages11
DOIs
Publication statusPublished - 2017

Keywords

  • Journal Article

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