What is it about?
For this study we worked around a cheap and selective way to do RNA sequencing of Leishmania. For this we updated the splice leader sequencing technique already published to make it work with Illumina sequencers but also to be able to multiplex, meaning to be able to sequence multiple samples on the same line of the sequencer. Then we compared it to the commercial golden standard from Illumina and found that our technique was performing the same as the commercial once. In a sequence time we compared both strategies on mixed samples with human DNA and leishmania in similar ratios as in clinical samples. In the particular case of a clinical sample we can not have access to only Leishmania RNA, we have to extract everything. The biggest issue is that human cells contain much more RNA than Leishmania cells so we have to sequence very deep to get the information on the parasite. We demonstrated performing in vitro infection assays that our technique was able to enrich up to 44% in Leishmania RNA, which is enough to study Leishmania transcriptome.
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Why is it important?
Leishmania is human pathogen part of the NTDs (Neglected Tropical Diseases) and is mostly studied in vitro. With this cheap and easy technique we can have fast and simple access to Leishmania gene expression inside clinical samples but also inside sandfly samples. Furthermore, every Trypanosomatid species have a splice leader to maturate their RNA but also cestodes, cnidaria and nematodes. So this techniques open the field of in situ transcriptome studies that are much more relevant than in vitro studies.
Perspectives
In previous studies we demonstrated correlation between genome structure (karyotype and local copy number variation) and transcript level. In comparison to DNA, RNA is much more abundant and would give access to the same information. So using the SL-RNAseq we could easily have access to part of the genome information, at least the coding part as well as the karyotype and the presence of local copy number variation. In the study we also demonstrate the power and the use of combining Illumina and SL-RNAseq on the same sample to get access to the host cell and the parasite transcriptome at the same time without performing heavy biological experiment that might affect the response.
Dr Franck Dumetz
University of Maryland
Read the Original
This page is a summary of: Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids
, Scientific Reports, June 2017, Nature,
DOI: 10.1038/s41598-017-03987-0.
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