RNA-seq generates new insights into Leishmania differentiation
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Leishmania donovani, an intracellular parasitic trypanosomatid, causes kala-azar, a fatal form of visceral leishmaniasis in humans. Infection occurs through a cycle whereby parasites (promastigote stage) living in the midguts of female sand flies are transferred to the host via a bite from an infected female sand fly, are phagocytosed by human macrophages, and are then transferred to phagolysosomes of human macrophages (amastigote stage). With the large increase in data that is generated by large-scale next-generation sequencing experiments, we embarked upon a systematic organization of the Myler lab next-generation sequencing data. This was necessary as using spreadsheets to track data had become impractical due to the long-term nature of several of the experiments, in addition to the turnover in lab personnel. We developed standard terminology and nomenclature, both key for ensuring consistency of organization among the various personnel involved in these experiments (biologists, bioinformaticians, and collaborators). We also developed two different data organization systems: one for the organization of raw data and one for the organization of analyzed data. Finally, we created a webpage to document the Myler lab data organization system and serve as a resource for those who are storing or searching for the data. Previous studies have demonstrated that L. donovani differentiation is regulated by changes in gene expression. Thus, we performed high throughput spliced leader RNA-sequencing to elucidate changes in transcript abundance for all cellular mRNAs during Leishmania donovani differentiation from promastigotes into amastigotes. Analyses revealed 534 statistically significant (p-value < 0.05 and mean log2 fold-change ≥ 1 or ≤ -1) genes and K-medians clustering of these genes revealed at least 6 different gene expression patterns (up early, late, or transiently; down early, late, or transiently). We also identified genes which encoded proteins (e.g. putative paraflagellar rod protein 1D, glucose transporter 2) that were expected or likely to be differentially expressed during promastigote-to-amastigote differentiation due to the morphological and environmental changes the parasite experiences during this process. In addition, it appears that the technique we have employed, spliced leader RNA-sequencing, allows us to detect gene expression changes in specific members of gene families, in contrast to the microarray studies we previously undertook, as well as providing insight into how post-transcriptional and post-translational regulation may have a role in mRNA expression changes during differentiation. Finally, this technique provides additional information about the sequences present in the 5’ untranslated regions of genes that can be used to improve genome annotation and may have a role in the generation of alternative transcripts.