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.

Variant antigen diversity inTrypanosoma vivaxis not driven by recombination

“…RNA was extracted using the RNeasy Mini Kit (Qiagen, UK) according to the manufacturer’s protocol, yielding a total RNA output between 117 ng and 13 μg per sample, quantified on the NanoDrop 2000 (ThermoFisher Scientific, Brazil). Up to 1 μg of total RNA was used to prepare multiplexed cDNA libraries as described 57 using the T. vivax splice-leader (SL) sequence 58 as the second cDNA strand primer. For samples up to day 30 p.i., the protocol of Cuypers et al (2017) 57 was followed exactly as described, quantified using Qubit HS dsDNA (Invitrogen, UK) and the Agilent 2100 Bioanalyzer (Agilent Technologies, UK), and sequenced at Centre of Genomic Research (Liverpool, UK) on a single lane of the HiSeq 4000 platform (Illumina Inc, USA) as 150 paired ends, producing 280M mappable reads.…”

“…Up to 1 μg of total RNA was used to prepare multiplexed cDNA libraries as described 57 using the T. vivax splice-leader (SL) sequence 58 as the second cDNA strand primer. For samples up to day 30 p.i., the protocol of Cuypers et al (2017) 57 was followed exactly as described, quantified using Qubit HS dsDNA (Invitrogen, UK) and the Agilent 2100 Bioanalyzer (Agilent Technologies, UK), and sequenced at Centre of Genomic Research (Liverpool, UK) on a single lane of the HiSeq 4000 platform (Illumina Inc, USA) as 150 paired ends, producing 280M mappable reads. However, as the library insert sizes produced were longer that recommended for the HiSeq 4000 platform (Illumina Inc, USA), the protocol for samples from days 30-45 p.i.…”

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

“…A variety of approaches therefore aim to obtain genome-wide SNP information without first performing pathogen isolation and culturing steps. Some studies separate target sequences from total DNA or RNA by exploiting base modifications or transcriptional properties specific to the pathogen 15 , vector 16 or host 17,18 . Others describe the use of biotinylated hybridization probes 19–22 or selective whole-genome amplification, e.g., based on the strand displacement function of phi29 DNA polymerase 23 .…”

SLFinder, a pipeline for the novel identification of splice-leader sequences: a good enough solution for a complex problem

“…This does not only allow to identify the presence of the mechanism in the group but having these sequences opens the possibility to use methodologies tailored toward SL Trans-Spliced transcripts. For example, "SL Trapping" [27] or "SL-seq" [28], both modified Next Generation Sequencing (NGS) protocols, allow an enriched sequencing of SL trans-spliced transcripts (e.g. [11]).…”