Use of the Oxford Nanopore platform to obtain maxicircles (kDNA) of Colombian isolates of Leishmania (Viannia) braziliensis for genomic and evolutionary studies.

Leishmania (Viannia) braziliensis is one of the main responsible of cutaneous leishmaniasis in Colombia. Its mitochondrial genome consists of maxicircles and minicircles. The maxicircles of all kinetoplastid flagellates are functional analogs of the mitochondrial genome of other eukaryotes. They consist of two distinct parts, referred as the coding region and the divergent region. The divergent region is composed of highly repetitive sequences and are the least explored segment of the mitochondrial genome of trypanosomatids, which makes their sequencing and assembly difficult, and for this reason there is a low report of complete maxicircle sequences available, as has been the case for organisms of the genus Leishmania. Therefore, the aim of this work is to show the obtaining of maxicircles from 7 clinical isolates of L. braziliensis through sequences from Oxford Nanopore Technology and their comparison with other trypanosomatids. The seven clinical isolates were assembled de novo using NGSEP and NECAT software, to confirm the identity of the clinical isolates as Leishmania braziliensis, a BLASTn was performed using the 7 assemblies obtained and the kinetoplast of a L. braziliensis strain as a reference (Access number: MN904516.1). In total 10 maxicircles were obtained from the de novo assembly and were verified by BLAST and%GC using the ribosomal genes 12S rRNA and 9S rRNA of L. peruviana since they are phylogenetically closely related (Accession number: BK010881.1). As for the annotation of the assembled maxicircles, the L. panamensis maxicircle was used as reference genome (Accession number: MK570510.1). And for the evaluation of nucleotide identity the annotated maxicircle was used in conjunction with other trypanosomatids of medical importance through the MUMmer software. A total of 10 maxicircles were obtained in the 7 assembled isolates with a size range of 24,300 to 31,000 bp and an average size of 27,400 bp. The size of the coding region comprised a total of 14,909 bp and the 10 assemblies had an average variable region size of 12,119 bp. Using bioinformatics tools, a circular maxicircle genome with a size of 27,737 bp was obtained, for the semi-automated annotation process were annotated 17 genes in the conserved region corresponding to the mitochondrial electron transport chain and 2 genes corresponding to ribosomal rRNAs. And finally, the nucleotide identity analysis of this maxicircle showed the following identities in the next order: L. peruviana with 99.02%, L. guyanensis with 98.03%, L. panamensis with 97.65%, L. infantum with 84.65%, L. tarentolae with 84.38%, L. donovani with 84.30%, L. major with 83.33%, L. mexicana with 83.28%, T. cruzi with 78.11% and finally T. brucei with 77.30%. The results of this work revealed that mitochondrial genomes can be obtained by means of ONT long reads with high quality and low cost, which allow the study of genomic variability and parasite biology and adaptive processes. [ABSTRACT FROM AUTHOR]