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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 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.

journal_name

Sci Rep

journal_title

Scientific reports

authors

Cuypers B,Domagalska MA,Meysman P,Muylder G,Vanaerschot M,Imamura H,Dumetz F,Verdonckt TW,Myler PJ,Ramasamy G,Laukens K,Dujardin JC

doi

10.1038/s41598-017-03987-0

subject

Has Abstract

pub_date

2017-06-16 00:00:00

pages

3725

issue

1

issn

2045-2322

pii

10.1038/s41598-017-03987-0

journal_volume

7

pub_type

杂志文章

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