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This protocol describes a stepwise process to identify proteins of interest from a query proteome derived from NGS data. We implemented this protocol on Moringa oleifera transcriptome to identify proteins involved in secondary metabolite and vitamin biosynthesis and ion transport. This knowledge-driven protocol identifies proteins using an integrated approach involving sensitive sequence search and evolutionary relationships. We make use of functionally important residues (FIR) specific for the query protein family identified through its homologous sequences and literature. We screen protein hits based on the clustering with true homologues through phylogenetic tree reconstruction complemented with the FIR mapping. The protocol was validated for the protein hits through qRT-PCR and transcriptome quantification. Our protocol demonstrated a higher specificity as compared to other methods, particularly in distinguishing cross-family hits. This protocol was effective in transcriptome data analysis of M. oleifera as described in Pasha et al.•Knowledge-driven protocol to identify secondary metabolite synthesizing protein in a highly specific manner.•Use of functionally important residues for screening of true hits.•Beneficial for metabolite pathway reconstruction in any (species, metagenomics) NGS data., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors. Published by Elsevier B.V.)

In this paper, we present the data acquired during transcriptome analysis of the plant Moringa oleifera [1] from five different tissues (root, stem, leaf, flower and seed) by RNA sequencing. A total of 271 million reads were assembled with an N50 of 2094 bp. The combined transcriptome was assessed for transcript abundance across five tissues. The protein coding genes identified from the transcripts were annotated and used for orthology analysis. Further, enzymes involved in the biosynthesis of select medicinally important secondary metabolites, vitamins and ion transporters were identified and their expression levels across tissues were examined. The data generated by RNA sequencing has been deposited to NCBI public repository under the accession number PRJNA394193 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA394193)., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Moringa oleifera is a plant well-known for its nutrition value, drought resistance and medicinal properties. cDNA libraries from five different tissues (leaf, root, stem, seed and flower) of M. oleifera cultivar Bhagya were generated and sequenced. We developed a bioinformatics pipeline to assemble transcriptome, along with the previously published M. oleifera genome, to predict 17,148 gene models. Few candidate genes related to biosynthesis of secondary metabolites, vitamins and ion transporters were identified. Expressions were further confirmed by real-time quantitative PCR experiments for few promising leads. Quantitative estimation of metabolites, as well as elemental analysis, was also carried out to support our observations. Enzymes in the biosynthesis of vitamins and metabolites like quercetin and kaempferol are highly expressed in leaves, flowers and seeds. The expression of iron transporters and calcium storage proteins were observed in root and leaves. In general, leaves retain the highest amount of small molecules of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Leishmaniasis is a neglected tropical disease caused by the pathogenic protozoan Leishmania donovani and it is transmitted by an infected sand fly. Approximately 0.4 million cases of Visceral Leishmaniasis are reported across the globe every year, of which 67% is from the Indian subcontinent. The currently available drugs have not been effective owing to their high toxicity levels, inadequate specificity, drug resistance, extended treatment periods and/or prohibitive prices. For this reason, hypothetical proteins in this pathogen, which constitute about 67% of its proteome, must be distinctly characterized and studied for their potential role as drug targets for Leishmaniasis. Domain information from PFAM and functional information from GO has been used to assign putative functions to 36 hypothetical membrane proteins in this protozoan. Furthermore, as a case study, we have performed a thorough sequence level characterization of a hypothetical protein E9BPD7 from the BT1 family of membrane proteins that transports folate/biopterin. Phylogenetic analyses of E9BPD7 have revealed interesting evolutionary correlations to BT1 family and MFS superfamily, which have significant roles in a number of diseases and drug resistance pathways., (© 2019 Biomedical Informatics.)

Leishmaniasis is a parasitic disease caused by the protozoan Leishmania, which is active in two broad forms namely, Visceral Leishmaniasis (VL or Kala Azar) and Cutaneous Leishmaniasis (CL). The disease is most prevalent in the tropical regions and poses a threat to over 70 countries across the globe. About 200 million people are estimated to be at risk of developing VL in the Indian subcontinent, and this refers to around 67% of the global VL disease burden. The Indian state of Bihar alone accounts for 50% of the total VL cases. While no vaccination exists, several pentavalent antimonials and drugs like Paromomycin, Amphotericin, Miltefosine etc. are used in the treatment of Leishmaniasis. However, due to their low efficacies and the resistance developed by the bug to these medications, there is an urgent need to look into newer species specific targets. The proteome information available suggests that among the 7960 proteins in Leishmania donavani, a staggering 65% remains classified as a hypothetical uncharacterized set. In this background, we have attempted to assign probable functions to these hypothetical sequences present in this parasite, to explore their plausible roles as druggable receptors. Thus, putative functions have been defined to 105 hypothetical proteins, which exhibited a GO term correlation and PFAM domain coverage of more than 50% over the query sequence length. Of these, 27 sequences were found to be associated with a reference pathway in KEGG as well. Further, using homology approaches, four pathways viz., Ubiquinone biosynthesis, Fatty acid elongation in Mitochondria, Fatty Acid Elongation in ER and Seleno-cysteine Metabolism have been reconstructed. In addition, 7 new putative essential genes have been mined with the help of Eukaryotic Database of Essential Genes (DEG). All these information related to pathways and essential genes indeed show promise for exploiting the select molecules as potential therapeutic targets.