Multiplexed Spliced-Leader Sequencing – A high-throughput, selective method for RNA-seq

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 a new multiplexed protocol for high-throughput, selective RNA-sequencing called SL-seq, developed by researchers at the Institute of Tropical Medicine, Antwerp.

The researchers 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.

Graphical summary of the SL-seq protocol

(A) cDNA generation with Superscript III and a primer that is partially random (7 nucleotides, grey), and partially fixed (yellow). Consequent degradation of the RNA strand with RNAse H, leaving a single stranded DNA molecule. (B) Second strand synthesis of SL-containing DNA molecules with Klenow fragment and a primer that is complementary with the SL (dark blue). (C + D) PCR for amplification and addition of adapter motives (red and purple) making the library compatible with the Nextera XT index kit (Illumina). (E + F) Final PCR adapter extension, indexing (orange and light blue) and amplification of the library fragments with the primers of the Nextera XT index kit. Dark Blue: SL/ complementary with SL, light grey: RNA, dark grey: DNA, green: poly-A tail. Other colors: primer and adapters sequences.