Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids

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 Splic...

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Autores principales:	Bart Cuypers, Malgorzata A. 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
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/42bf4dbddfc34561be9f8377912ded62
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spelling	oai:doaj.org-article:42bf4dbddfc34561be9f8377912ded622021-12-02T12:30:11ZMultiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids10.1038/s41598-017-03987-02045-2322https://doaj.org/article/42bf4dbddfc34561be9f8377912ded622017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03987-0https://doaj.org/toc/2045-2322Abstract 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.Bart CuypersMalgorzata A. DomagalskaPieter MeysmanGéraldine de MuylderManu VanaerschotHideo ImamuraFranck DumetzThomas Wolf VerdoncktPeter J. MylerGowthaman RamasamyKris LaukensJean-Claude DujardinNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Bart Cuypers Malgorzata A. 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 Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids
description	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.
format	article
author	Bart Cuypers Malgorzata A. 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
author_facet	Bart Cuypers Malgorzata A. 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
author_sort	Bart Cuypers
title	Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids
title_short	Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids
title_full	Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids
title_fullStr	Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids
title_full_unstemmed	Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids
title_sort	multiplexed spliced-leader sequencing: a high-throughput, selective method for rna-seq in trypanosomatids
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/42bf4dbddfc34561be9f8377912ded62
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Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids

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