Resequencing of the Leishmania infantum (strain JPCM5) genome and de novo assembly into 36 contigs

Abstract

Leishmania parasites are the causative of leishmaniasis, a group of potentially fatal human diseases. Control strategies for leishmaniasis can be enhanced by genome based investigations. The publication in 2005 of the Leishmania major genome sequence, and two years later the genomes for the species Leishmania braziliensis and Leishmania infantum were major milestones. Since then, the L. infantum genome, although highly fragmented and incomplete, has been used widely as the reference genome to address whole transcriptomics and proteomics studies. Here, we report the sequencing of the L. infantum genome by two NGS methodologies and, as a result, the complete genome assembly on 36 contigs (chromosomes). Regarding the present L. infantum genome-draft, 495 new genes have been annotated, a hundred have been corrected and 75 previous annotated genes have been discontinued. These changes are not only the result of an increase in the genome size, but a significant contribution derives from the existence of a large number of incorrectly assembled regions in current chromosomal scaffolds. Furthermore, an improved assembly of tandemly repeated genes has been obtained. All these analyses support that the de novo assembled L. infantum genome represents a robust assembly and should replace the currently available in the databases.

The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations

“…Peptide identification from raw data was carried out using the PEAKS Studio X search engine (Bioinformatics Solutions Inc, Waterloo, ON, Canada). A custom Python script was used to create a database comprising all possible open reading frames (ORF) coding for protein sequences of ≥20 amino acids existing in any of the six-frames in the L. infantum JPCM5 strain genome sequence [13]. This database (named LINF-all-ORFs) consisted of 294,654 entries.…”

“…From the MS/MS data, it could be seen that only those associated with peptides longer than seven amino acids were considered for protein identification. Among the identified proteins, 2344 proteins matched with previously annotated proteins [13]. Moreover, eight novel proteins were uncovered, thus legitimating the search strategy.…”

“…The main objective of this study was to obtain the experimental proteome of the L. infantum promastigote stage with the additional aim of improving current genomic annotations. For this purpose, a recently published re-sequenced genome [13] was used. However, we did not restrict the search of peptide spectra on currently annotated protein-coding genes; instead, a database consisting of all possible polypeptides (equal or larger than 20 amino acids) was used (see Materials and Methods for further details).…”

“…Currently, 3482 out of 8590 annotated proteins (around 40%) in the L. infantum genome have the status of hypothetical proteins; the MS spectra obtained in this work provided experimental evidence of the real existence for 456 of those hypothetical proteins. The experimental MS data were searched against the UniProt protein database and a database consisting of all possible polypeptides encoded in the six-frames of the L. infantum genome (based on v2/2018; www.leish-esp.cbm.uam.es; [13]).…”

“…The Venn diagram was created by the tool available at bioinformatics.psb.ugent.be/webtools/. Note-the discrepancy between the number of proteins identified by Rosenzweig et al [20] (see text) and those represented in the Venn diagram (1713 vs. 1708) was due to 5 gene duplications that were corrected after re-assembling of the L. infantum genome [13].…”