Resequencing and de novo Assembly of Leishmania (Viannia) guyanensis from Amazon region: Genome assessment, Aneuploidies, and Phylogenetic insights

Resequencing and de novo Assembly of Leishmania (Viannia) guyanensis from Amazon region: Genome assessment, Aneuploidies, and Phylogenetic insights | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Resequencing and de novo Assembly of Leishmania (Viannia) guyanensis from Amazon region: Genome assessment, Aneuploidies, and Phylogenetic insights Lucas George Assunção Costa, Edivaldo Costa Sousa Junior, Camila Cristina Cardoso Gomes, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7303461/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Leishmania genus includes 20 human pathogenic species, 15 occurring in the Americas. In Amazonia, Leishmania guyanensis is frequently associated with therapeutic failure, what is related to genetic plasticity and adaptability during drug exposure. The genomic studies of this species are important to understand different aspects of parasite biology including genetic adaptability to different environments and possible therapeutic alternatives. Taking this into account, we sequenced a L. guyanensis strain (MHOM/BR/75/M4147), performed the quality assessment of the reads (FASTP), and the genome assembly (MEGAHIT). The taxonomic classification was accomplished using BLASTn and Kraken2 software, and the annotation of Leishmania contigs using Augustus v.3.5. The assembled genome showed a size of 31 Mb, N50 = 4.743 bp, genome coverage of 40.85x, and 99,5% of its contigs were related to Leishmania . We confirmed the presence of 36,665 SNPs, 8210 Indels, and aneuploidies. After annotation, we identified 3119 proteins associated with well-known molecular functions in L. guyanensis and 6.371 ortholog genes shared by L. guyanensis , L. major , and L. panamensis . The phylogenetic analysis using the polA1 gene correctly grouped L. guyanensis showing discriminatory potential to identify all Leishmania species, highlighting L. martiniquensis as the more divergent species within the Leishmania genus. The overall results complement the extant genomic data of L. guyanensis and encourage progress in the species-specific diagnostic of Leishmania spp. Biological sciences/Computational biology and bioinformatics Biological sciences/Evolution Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology Leishmania guyanensis genome assembly genomic annotation polA1 phylogeny Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The Leishmania genus (Kinetoplastida; Trypanosomatidae) includes species responsible for a spectrum of tropical zooanthroponotic diseases known as leishmaniasis. The common clinical manifestations of the disease are cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (ML), and visceral leishmaniasis (VL). The protozoans of this genus are transmitted to mammal reservoirs, including humans, by the bite of several phlebotomous species (Psychodidade: Phlebotominae), which are broadly spread in forest areas 1 , 2 , 3 . The diverse species of the Leishmania genus cluster in four subgenera: Leishmania ( Viannia ), Leishmania ( Leishmania ), Leishmania ( Sauroleishmania ), and Leishmania ( Mundinia ). The first subgenus includes Leishmania pathogens broadly distributed in the Americas. Nearly fifty Leishmania species are currently known; twenty are human pathogens, including 15 endemics in the Americas 4 , 5 . The Leishmania ( Viannia ) subgenus is implicated in most CL and ML cases reported in Latin America 6 . Consequently, it is mandatory to carry out studies on the comparative genomics of the species housed in this subgenus that may reveal differences useful to explain how biological traits of Leishmania Viannia parasites impact medically relevant characteristics such as drug resistance, clinical outcome, virulence, and pathogenicity. Formerly, CL and ML complications were commonly associated with L. ( V. ) braziliensis , the most disseminated species in the Americas. However, it is currently known that L. ( V. ) guyanensis causes not only single skin lesions (or multiple lesions at determined frequency) but also nasopharyngeal mucosa destruction in more severe cases 7 , 8 . This species occurs in several Latin American countries such as Panama, Brazil, Bolivia, Colombia, French Guiana, Suriname, Venezuela, Ecuador, and Argentina 9 , 10 , 11 . Leishmania ( Viannia ) guyanensis transmission is strongly related to human displacement through heavy vegetation where the presence of mammal species such as Choleopus didactylus , Tamandua tetradactyla , and Didelphis marsupialis , natural reservoirs of this parasite, occur. In Brazil, the L. ( V. ) guyanensis distribution is especially broad in the Amazon region, where drug failure rates are high, and a different treatment choice is mandatory due to the predominance of this Leishmania species 12 , 13 . Under pressure, L. ( V. ) guyanensis develops adaptative mechanisms capable of inducing drug uptake/efflux modifications in the first-line therapy used to treat CL, which causes treatment failure 14 , 15 . These and other biological characteristics, including virulence, pathogenicity, proliferative capacity, and immune modulation, are directly related to pathogens' genomes 16 . Genomic plasticity is a common feature of the Leishmania genus, including aneuploidy and chromosomal fission/fusion events. Moreover, the kinetoplastid genome organization shows genes grouped in polycistronic transcription units and an evident scarcity of introns in their genes. This fact evidences a basal genomic structure without most of the more complex modular genetic elements found in other eukaryotes 17 , 18 . The L. ( V. ) guyanensis genome has an average size of 32 Mb containing nearly 8000 protein-coding genes and 35 chromosomes due to the fusion of chromosomes 20 and 34 16 . When thoroughly described, this genomic data represents a source of accurate and robust information necessary for understanding parasite adaptability and survival strategies that might contribute to developing efficient therapeutic interventions. In the middle of several initiatives aimed at sequencing genomes of parasites from the Leishmania genus 19 , 16 , 20 , this study describes the genome of a L . ( V. ) guyanensis strain from Amazonia, a Leishmania species poorly investigated 21 . Our study is intended to elucidate not only the genetic architecture and the phylogenetic status of L . ( V. ) guyanensis but also to offer further insights into the genomics of this parasite that represent a baseline to improve the diagnostic and treatment of CL and ML in the Americas. Material and methods Ethics This study does not involve human subjects or tissue samples but only Leishmania promastigotes and computational analyses, therefore ethical approval is not required. DNA extraction and sequencing The Leishmania guyanensis strain was provided by a Leishmania Collection (CLIOC) that is part of the Network of Biological Resource Centers for Conformity Assessment of Biological Material, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. The genomic DNA of the L. ( V. ) guyanensis strain (MHOM/BR/75/M4147) was isolated using the Wizard®ฏ Genomic DNA Purification kit (Promega) following the manufacturer's instructions, rehydrating the whole DNA in a final volume of 50 µL. The genomic library of this parasite was then prepared using the Nextera XT DNA library preparation kit (Illumina, Inc.) following the manufacturer's protocols to subsequently be sequenced by the NexSeq 500 (Illumina, Inc.) platform using the NextSeq 500/550 High Output Kit v2.5 (300 cycles) and a pared-end sequencing approach. Reads Quality assessment To get rid of adapters and low-quality reads (Phred < 20) after the sequencing process, we used the Fastp software 22 and the software FastQC 23 to depict read-quality data. Genome Assembly We used a de novo assembly approach employing no reference genome to assemble all reads in contigs using the MEGAHIT v1.2.9 assembly software 24 . To create scaffolds from the assembled contigs, we used the software SSPACE v.3.0 25 . Taxonomical Classification of Reads and Contigs To perform the taxonomical classification of reads and contigs obtained, we first infer the sequence homology of these sequences by a local alignment with sequences from a local curate Leishmania DNA database using the Blastn algorithm 26 . After sequence homology determination, we inferred the taxonomical status of reads and contigs by performing a classification based on k-mer and adding taxonomical tags to each sequence using Kraken2 software 27 . This classification allowed us to extract all contigs belonging to Leishmania to perform the genomic annotation of L. ( V. ) guyanensis (MHOM/BR/75/M4147) subsequently. Genome Annotation The genomic annotation of this parasite was performed using the AUGUSTUS software v.3.5, using as a reference the genomic structure of Leishmania ( Sauroleishmania ) tarentolae. We constructed a local genomic database of 69 Leishmania genomes (Supplementary Table 1) retrieved from GenBank to compare genomic annotations and analyze and correct open reading frames (ORFs) positions and structures. In this way, we provided more accurate evidence of gene function during the gene annotation of this parasite. To depict the genome and manually curate ORFs obtained during the annotation process, we used the bioinformatics suite Geneious v.8.1.4 28 . The manual curation was finished when we analyzed the base quality and integrity of each ORFs. Gene Orthologs Assessment The genomic data of L. ( V. ) guyanensis (MHOM/BR/75/M4147) was used to perform a Pangenome analysis aimed at identifying orthologs genes comparing this genome to the genome assembly of Leishmania ( V. ) shawi (GCA_962240455), Leishmania ( V. ) panamensis (GFC_000755165.1) and Leishmania ( L. ) major (GCF_000002725.2). To do so, we used the software BLASTp v2.5.0 29 , Get_Homologues 30 , and the statistic software R using the Venn library 31 . Mapping to reference genome, Genome Coverage, and Variant Call The mapping to the reference genome of L. ( V. ) guyanensis was performed using Bowtie2 32 , allowing an assessment of chromosomic coverage. This read mapping also allowed us to determine chromosome ploidy (chromosome copy number for each genome) by dividing each chromosome coverage by half of each thirty-five chromosome's average coverage and assuming a diploid organism. Single nucleotide polymorphisms (SNPs) and insertion/deletion (indels) events were identified using BCFtools v1.2 33 to detect nucleotide differences between the resequencing strain of L. ( V. ) guyanensis (MHOM/BR/75/M4147) and the reference genome of this species (GenBank CP103914-CP103949). Phylogenetic inference The DNA polymerase alpha catalytic subunit (polA1) was selected as the best molecular target to infer Leishmania phylogeny after evaluating its phylogenetical signal with the TREE-PUZZEL v.5.3 algorithm 34 using the likelihood-mapping method. To assess the phylogenetic signal, we used 69 Leishmania genomes (Supplementary Table 1). The polA1 gene from these genomes was identified and extracted using the software Geneious v8.1.4 to perform a phylogenetic analysis using the maximum likelihood method (ML) included in the IQTREE2 software 35 after selecting the best nucleotide substitution model by the algorithm of the software. The software parameters were set up to find the more likely tree topology testing each clade's robustness by bootstrap analysis. The choice of the best phylogenetic tree considered the genetic identity threshold among Leishmania species inferred by a sequence identity matrix constructed by the software. All software parameters were set to standard conditions unless another configuration was required. Results Genome Assembly The genome sequencing of the L. (V.) guyanensis (MHOM/BR/75/M4147) produced 9.847.950 reads with an average size of 151 pb. After filtering low-quality reads and excluding adapters and artifacts from the sequencing process, we obtained 8.839.174 high-quality reads, a reduction of 10%. The de novo assembly produced 14.097 contigs with an N50 value of 4.743 bp, an average contig size of 2.239 bp, and an average genome coverage of 40.85x. The number of scaffolds constructed was identical to the number of contigs (14.097). The genome size of the L. (V.) guyanensis was 31.565.639 pb (31 Mb) with a G + C content of 57.44%. Consequently, we obtained a new complete genome assembly (whole genome shotgun) for the Leishmania isolate analyzed herein. Table 1 gathers all the genome assembling results obtained in this study and the ones produced in earlier Leishmania genomic studies. The genome assembly for L. (V.) guyanensis (MHOM/BR/75/M4147) was deposited in the GenBank database under the accession number GCA_051201275.1. The data is publicly available at https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_051201275.1/ . Table 1 Assembled Genomes of Leishmania spp. Assembled genomes of Leishmania spp Specie L. guyanensis L. guyanensis (M4147) L. guyanensis (LgCL085) L. naiffi L. panamensis (PSC-1) L. braziliensis L. infantum L. major (M4147) (LnCL233) (M2904) (JPCM5) Friedlin Strain Contigs 14,097 35 10,308 14,682 N/A N/A N/A 36 N50 contigs (Kb) 4743 1,1 9.6 5.7 N/A N/A N/A N/A Scaffolds 14,097 35 2,8 6,53 108 N/A N/A N/A N50 scaffold (Kb) 4743 1,1 95.4 24.3 674 N/A N/A N/A Number of Gaps 0 0 1,557 3,853 553 876 440 N/A Average Coverage 40.85x 27x 56x 36x 30x N/A N/A N/A Genome Size (Mb) 31.56 32.5 31.01 30.34 30.69 31.24 31.92 32.8 G + C content (%) 57,44 58 N/A N/A 57.56 57.72 59.58 59.7 Protein-coding Genes 7192 N/A 8,23 8,104 7,748 8,175 8,199 8,272 Total Number of Genes 7698 N/A 8,376 8,262 7,933 8,37 8,26 8,341 Reference Current study Genbank Coughlan Coughlan Llanes Llanes Llanes Ivens et al. (2018) et al. (2018) et al . (2015) et al . (2015) et al . (2015) et al . (2005) Taxonomical Assessment Most of the contigs produced in this study (99.95%, 14.090 contigs) were found to be related to the Leishmania genus. All selected contigs showed an average nucleotide identity value of 96.29% with Leishmania spp . The contigs tagged as belonging to Leishmania spp. were then used in the genomic annotation process. Variants Call and Chromosome Coverage We searched for variants and identified 36.665 SNPs distributed through the 35 L. ( V. ) guyanensis chromosomes and 8.210 indels (Supplementary Table 2). R eads mapping to the L. ( V. ) guyanensis reference genome (GenBank CP103914-CP103949) identified chromosomes with coverage values between 41x and 111x (Supplementary Table 2). Additionally, there was great heterogeneity in the chromosome ploidy (values between 1.2 and 3.4), highlighting chromosomes 2 and 8 that presented a condition close to trisomy. The ploidy variations throughout L. ( V. ) guyanensis chromosomes are depicted in Fig. 1 . Genomic Annotation The gene annotation of the L. ( V. ) guyanensis (MHOM/BR/75/M4147) revealed a lack of introns and allowed us to identify 7.698 genes, 99.9% (7.192) of which were associated with known molecular functions. After analyzing all the annotated data, we identified 3.119 different proteins related to the metabolic pathways of the parasite. The proteins with more associated genes were Calpain-like cysteine peptidase (155), protein kinase domain-containing protein (114), and transmembrane protein (75). Figure 2 depicts the main 20 relevant proteins identified during the annotation process. Gene Orthologs Analysis The gene annotation of L. ( V. ) guyanensis (MHOM/BR/75/M4147) allowed us to study the Pangenome, orthologs genes, and core genome that altogether represent the entire group of annotated genes extant in a particular taxonomic group (MEDINI et al ., 2020). Therefore, we identified 6.371 genes shared by L. ( V. ) guyanensis , L. ( V. ) panamensis , L. ( V. ) shawi , and L. ( L. ) major . Fourteen of these genes were exclusive of L. ( V. ) panamensis , 13 of L. ( V. ) guyanensis , 23 of L. ( V. ) shawi , and 299 of L . ( L. ) major . The Pangenome of these four Leishmania species encompasses 7.791 genes. Regarding the Leishmania species core genome, each species showed a characteristic genetic expansion (expansion in orthologs’ copy numbers). For example, we found 22 expanded genes in L. ( V. ) guyanensis MHOM/BR/75/M4147 (Supplementary material 3). The Pangenome analysis result is depicted in Fig. 3 . Phylogenetic Inference As a result of the phylogenetic analysis, the four Leishmania subgenera were grouped into independent monophyletic clusters supported by bootstrap values higher than 90%. Furthermore, all Leishmania complexes within each subgenus clustered into specific subclades supported by bootstrap values around 90% or higher. Regarding the sequence identity analyses, the species from the subgenera Leishmania ( Leishmania ), Leishmania ( Sauroleishmania ), Leishmania ( Viannia ), and Leishmania ( Mundinia ) showed sequence identity values of 90%, 99.8%, 96.8%, and 84.7%, respectively (Fig. 4 ). Discussion The genome assembling of L. ( V. ) guyanensis (MHOM/BR/75/M4147) performed herein strengthens the extant genomic data of this Leishmania species, providing excellent values of completeness and coherence when compared with the current L. ( V. ) guyanensis reference genome. An N50 value of 6.029 bp supported the good quality of the genome assembly as the parameter suggested that most of the contigs were long enough and well-assembled. The clear evidence that the scaffold number obtained was identical to the contigs number also supports the completeness of the assembled contigs, which are long enough and need no additional connections as links or bridges to create scaffolds. This fact reflects the good quality of the genome assembly obtained by the de novo method. Certainly, the quality filtering and taxonomical identification of the reads were critical for obtaining the correct genome structure since 9% of the reads were classified as sequencing artifacts. The reference genome of L. ( V. ) guyanensis (GenBank CP103914-CP103949) presented an average coverage value of 27.0x, while the coverage value obtained in this study for the same Leishmania strain was 40.85x. This higher coverage might have helped to acquire more accurate results in other genome-based analyses, such as SNPs identification and variant calls. Moreover, this study includes a robust genome annotation for this L. ( V. ) guyanensis strain, information that is still missing in genome databases such as TriTrypDB and GenBank. As the L. ( V. ) guyanensis genome assembly performed in this study, the assembly reported earlier for the L. ( V. ) guyanensis LgCL085 genome 36 presented good assembling parameters. However, a different bioinformatic workflow including assembly corrections, gap filling, and junction joining was necessary to complete the genome assembly. Both L. ( V. ) guyanensis (LgCL085) and L. ( V. ) panamensis (PSC-1) genomes available in the GenBank were characterized via de novo assembly 16 , 36 , representing the only strains from the L. ( V. ) guyanensis complex with complete genomes available in genome databases. The genomic characterization of L. ( V. ) guyanensis (MHOM/BR/75/M4147) performed in this study complements the genomic data of this species, providing indicative parameters of quality and completeness lacking in most genomic studies of Leishmania . Generally, the genome assembly of L. ( V. ) guyanensis (MHOM/BR/75/M4147) obtained herein showed similar genomic characteristics as the ones previously reported for L. ( V. ) braziliensis 37 , Leishmania ( Leishmania ) amazonensis 38 , and L. ( V. ) panamensis 16 , which present genome sizes of nearly 30Mb, average G + C content of 57%, and a value of predicted gene quantity close to 8000. This result indicates convergency among methods used to assemble Leishmania genomes, highlighting the importance of resequencing previously characterized Leishmania species, especially those with intrinsic epidemiological and clinical relevancy. The L. ( V. ) guyanensis genome obtained in this study presented chromosome number variation, a biological event known as aneuploidy 39 . Aneuploidy tolerance is a trypanosomatid ancestral characteristic that might be prevalent in a monophyletic clade that underwent no significant genomic reorganization 40 . This characteristic frequently occurs in the Leishmania genus without defined patterns among species, resulting in karyotype diversity within the parasite lineages (mosaic aneuploidy) 41 , 42 . The present analysis reveals that the L. ( V. ) guyanensis karyotype varies depending on the genomic reference used for gene mapping. We found that when the L. ( V. ) guyanensis genome is mapped to the L. ( V. ) braziliensis genome, chromosomes 8 and 31 seem to be supernumerary. Nonetheless, when the L. ( V. ) guyanensis genome is mapped to the reference genome for this species, the karyotype reveals a tendency to the disomic state, with chromosomes 1, 2, 3, 8, 31, 33, and 34 showing coverage values higher than 2.5. the intermediary values observed in the chromosomic coverage assessment might result from a mixed cellular population where chromosomic ploidy variation exists in the same sample, a common occurrence in Leishmania spp. 42 , 43 , 44 . A more significant deviation from the disomic state was observed during the read mapping in chromosomes 2 and 8. This fact also supports that the genomic reference choice influence significantly affects karyotype even among species belonging to the same subgenus, highlighting the importance of using a specific reference genome from the same species to perform more accurate analyses. Even though L. ( V ) guyanensis (MHOM/BR/75/M4147) was mapped to the reference genome of this species, it was found 36,665 SNPs in this Leishmania strain. This genetic variability might arise during parasite culture in different microenvironments, resulting in a genetic adaptation favoring parasite survival to various environmental stimuli 45 . The study carried out herein complements the genetic architecture previously described for L. ( V ) guyanensis (MHOM/BR/75/M4147) (reference) identifying genes (7726) and describing functional proteins. In this sense, the gene ontology analyses of the L. ( V. ) guyanensis (LgCL085) revealed the presence of 8230 protein-coding genes. Most of these proteins were associated with ortholog genes reported in the Viannia subgenus whose copy numbers vary from one species to another 36 . Due to the polycistronic nature of this genus, each protein is not strictly associated with one gene, and there are also no coding DNA and ORFs coding for more than one protein. This fact was verified after finding a high level of genes associated with one functional protein. The initial description of 3128 protein-coding genes in L. ( V. ) guyanensis might aid in disclosing the translational composition of this species, a fact of paramount importance for understanding its cellular and metabolic processes 37 . The gene annotation was of value to identify essential elements including start and stop codons, exon-intron junctions, protein-coding regions, and promoters and regulatory regions. As a result, we observed a lack of introns in most of the L. ( V. ) guyanensis protein-coding genes, evidence previously reported in genomic studies of Leishmania earlier 46 , and that could be linked to inherent polycistronic characteristics of the Leishmania genus. This polycistronic region includes a DNA region processed during transcription to produce a long polycistronic RNA including non-related genes organized into a genomic architecture resembling the prokaryotes' gene organization 47 , 48 . The workflow described herein identifies proteins and ortholog genes in microorganisms in general and has also been used earlier to assess Leishmania spp. genomes 36 . Using this strategy, we identified ortholog genes shared by L. ( V. ) guyanensis , L. ( V. ) panamensis , and L . ( L. ) major and a reduced group of genes (48) restricted to L. ( V. ) guyanensis . Among these three species, L. ( L. ) major showed the highest number of genes unrelated to the other species (1631), probably because it belongs to a different Leishmania subgenus. Conversely, we found fewer exclusive genes between L. ( V. ) panamensis and L. ( V. ) guyanensis . Generally, gene ortholog studies have indicated low quantities of species-specific genes in Leishmania 16 , 36 , 19 . Additionally, we observed a trend indicating that closely related Leishmania species from the same complex share more ortholog genes in the Pangenome than those Leishmania parasites from different subgenus. This might be explained by the great divergency bridge between Leishmania spp. from different subgenus due to the parasites' adaptation to conditions found in each transmission cycle. This biological adjustment of parasites from different Leishmania subgenera during the evolutionary process might explain the functional diversity found in the Leishmania genus nowadays. Indeed, the functional diversity produced in this way might account for the differential group of orthologs belonging to the different taxonomic units within the Leishmania spp . Pangenome. The phylogenetic analysis was important for validating the genome assembly data of L . ( V. ) guyanensis (MHOM/BR/75/M4147) because it might explain any discrepancies found during the assembly process 49 . The analysis using TREE-PUZZLE software showed a high phylogenetic signal of the polA1 gene favoring the construction of a well-supported phylogenetic tree. This molecular target has been used to identify Leishmania species 50 , 51 , showing to be a potential candidate to differentiate cryptic species such as L . ( V. ) guyanensis and L. ( V. ) panamensis , Leishmania species associated with tegumentary leishmaniasis in the Americas. This fact is important because, in the last 24 years, the validation of Leishmania species has been a matter of discussion. The main taxonomical problem lies in the initial Leishmania taxonomical proposal based mainly on the biological and eco-epidemiological characteristics of the current species 52 , 53 , 54 . The molecular marker polA1 was used earlier to distinguish and group Leishmania ( Mundinia ) martiniquensis by phylogenetics when the Multilocus Enzyme Electrophorese analyses (MEE) could not differentiate this Leishmania species 50 . In this study, the phylogenetic analysis using the polA1 gene clustered in different clades of cryptic species, some Leishmania species from the same complex, such as Leishmania ( Leishmania ) donovani and L. ( L. ) infantum; L. ( V. ) guyanensis and L. ( V. ) panamensis ; L. ( V. ) braziliensis and Leishmania ( Viannia ) peruviana. All sequences from L. ( V. ) guyanensis and L. ( V. ) panamensis species clustered into different monophyletic clades, unlike studies based on an HSP70-based approach that presented problems differentiating these cryptic species 55 . Phylogeny based on the HSP70 gene in Leishmania has been of help with the subgenera assignment and raised some important questions on the validity of some species belonging to the L. ( V. ) guyanensis complex 56 . The phylogenetic analysis performed herein showed the potential of the polA1 gene for the Leishmania complexes validation when compared with the Leishmania HSP70 gene region. The main pitfall reported in HSP70-based protocols is their problems discerning some species from the Viannia subgenus since the geographical distribution of the Leishmania species in the Americas has favored a high ancestral genetic flux 57 . Even using the Multilocus Sequence Typing approach (MLST), the phylogenetic differentiation between L. ( V. ) guyanensis and L. ( V. ) panamensis from the L. ( V. ) guyanensis complex is still problematic 36 . The advances in the etiological characterization of the Leishmania genus have suggested modifications in the taxonomic nomenclature of species 58 , 59 and the validation of complexes within subgenera 60 . To do so, it is preconized to use and look at DNA-sequence-based strategies instead of traditional methods based on isoenzymes, a recommendation we followed by using a molecular approach based on the PolA1 gene. The phylogenetic species concept is based on creating a monophyletic group with characteristics different from other groups and following a model of parental ancestry 61 . Consequently, the tree topology obtained herein using the polA1 gene from Leishmania species suggests the L. ( V. ) guyanensis species complex validation. Furthermore, the phylogenetic tree indicated nucleotide identity grades of at least 90% among species from the same subgenus except the Leishmania ( Mundinia ) subgenus (84.7%). This parameter indicates the number of identical sites among clade components. Observing the tree topology, it is evident that L. ( M. ) martiniquensis showed the highest phylogenetic distance when compared visually with the rest of the species from the Mundinia subgenus, therefore, influencing the global nucleotide identity value assessed for this group. Indeed, when removing the L. ( V. ) martiniquensis from the analysis, the Mundinia clade clustered species with a nucleotide identity value of 95%, an increasing rate of more than 10%. This result suggests that L. ( V. ) martiniquensis might be the most divergent species of the whole Leishmania genus. Conclusion The genomic assembly performed in this study showed equivalent results to the rest of the Leishmania assembly data obtained earlier complementing and improving the genomic architecture data of L. ( V. ) guyanensis. Further analysis such as variant calls, aneuploidy studies, and the genomic annotation performed herein add important information to the initial genomic description of this Leishmania species and describe relevant functional proteins important for Leishmania etiopathogenesis. The polyA1 marker proved to be a promising candidate for developing phylogenetic studies of Leishmania easily identifying Leishmania subgenera and complexes and showing outstanding discrimination power to validate and differentiate Leishmania species belonging to these two taxonomical categories. The phylogenetic analysis also allowed us to identify a high divergency level within the Leishmania ( Mundinia ) subgenus, highlighting L . ( M .) martiniquensis as probably the most divergent species in the Leishmania genus. Overall, this study validates the efficiency of modern methods based on sequencing, genomic analysis, and phylogeny in the Leishmania species characterization process. Lastly, this efficiency obtained by up-to-date methods facilitates understanding important molecular, biological, and epidemiological aspects of Leishmania spp. in the Americas paving the way for further valuable studies on leishmaniasis in this continent. Declarations Acknowledgments The authors acknowledge the following organizations in Brazil and Panama for supporting researchers and students involved in this study: Programa de Pós-Graduação Bionorte - Rede de Biodiversidade e Biotecnologia da Amazônia, Universidade Federal do Pará , Brazil; Programa Institucional de Bolsas de Iniciação Científica (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq), Instituto Evandro Chagas , Brazil; Instituto Conmemorativo Gorgas de Estudios de la Salud , Panama and Sistema Nacional de Investigación , Panama. Authors contributions L.G.A.C. wrote the initial manuscript draft, made formal analysis and generated figures. E.C.S.J. devised the computational strategy, did formal analysis and results interpretation, reviewed and edited the draft concerning bioinformatic aspects. C.C.C.G. analyzed the formal chromosomal ploidy, wrote respective results and formatted references. M.A.F.I. improved the formal genome assembling workflow and composed the table; F.S.A. reviewed the results analysis and interpretation; wrote and translated the final draft fitting to the journal format. L.M.G. Contributed to the conception, design and planning of the study, fundraising, results analysis and interpretation, final draft review and editing, supervised the research team and mentored all contributing students. All authors reviewed the manuscript. Funding Financier of studies and projects (FINEP), Brazil, agreement number 01.22.0495.00; Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil, within the scope of Notice n o . 13/2020 - Postgraduate Development Program - Process: 88887.919409/2023-00. Data availability Sequence data that support the findings of this study have been deposited in the GenBank database under the accession number GCA_051201275.1. The data is publicly available at https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_051201275.1/. Additional Information Competing interests The authors declare no competing interests. Correspondence and request for materials should be addressed to Lourdes Garcez Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral about jurisdictional claims in published maps and institutional affiliations. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. References Alencar, R. B., Justiniano, S. C. B. & Scarpassa, V. M. 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Phylogeny of Leishmania species based on the heat-shock protein 70 gene. Infect. Genet. Evol. 10 (2), 238–245. http://doi.org/10.1016/j.meegid.2009.11.007 (2010). Hoyos, J., Rosales-Chilama, M., León, C., González, C. & Gómez, M. A. Sequencing of hsp70 for discernment of species from the Leishmania (Viannia) guyanensis complex from endemic areas in Colombia. Parasit. Vectors . 15 (1), 406. http://doi.org/10.1186/s13071-022-05438-w (2022). Akhoundi, M. et al. Leishmania infections: Molecular targets and diagnosis. Mol. Aspects Med. 57 , 1–29. http://doi.org/10.1016/j.mam.2016.11.012 (2017). Maurício, I. L., Stothard, J. R. & Miles, M. A. The strange case of Leishmania chagasi . Parasitol. Today . 16 (5), 188–189. http://doi.org/10.1016/s0169-4758(00)01637-9 (2000). Lukes, J. et al. Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy. Proc. Natl. Acad. Sci. U S A . http://doi.org/10.1073/pnas.0703678104 (2007). Cupolillo, E., Grimaldi, G. Jr. & Momen, H. A. general classification of New World Leishmania using numerical zymotaxonomy. Am. J. Trop. Med. Hyg. 50 (3), 296–311. http://doi.org/10.4269/ajtmh.1994.50.296 (1994). Cupolillo, E., Boité, M. C. & Porrozzi, R. Considerações sobre taxonomia do gênero Leishmania . In: CONCEIÇÃO-SILVA, F.; ALVES, C. R. Leishmanioses do Continente Americano. 1ª ed. Rio de Janeiro (ed. Editora Fiocruz) 39–51, (2014). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7303461","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":498805779,"identity":"baab06e4-ab78-4596-a066-7b93e04aa6dd","order_by":0,"name":"Lucas George Assunção Costa","email":"","orcid":"","institution":"Instituto Evandro Chagas/Secretaria de Vigilância em Saúde e Ambiente/Ministério da Saúde","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"George Assunção","lastName":"Costa","suffix":""},{"id":498805780,"identity":"45d2db25-59c7-466d-b96f-c856351c6daa","order_by":1,"name":"Edivaldo Costa Sousa Junior","email":"","orcid":"","institution":"Instituto Evandro Chagas/Secretaria de Vigilância em Saúde e Ambiente/Ministério da Saúde","correspondingAuthor":false,"prefix":"","firstName":"Edivaldo","middleName":"Costa Sousa","lastName":"Junior","suffix":""},{"id":498805781,"identity":"9dce88f9-a899-4061-83cb-c7fb66ada5bc","order_by":2,"name":"Camila Cristina Cardoso Gomes","email":"","orcid":"","institution":"Instituto Evandro Chagas/Secretaria de Vigilância em Saúde e Ambiente/Ministério da Saúde","correspondingAuthor":false,"prefix":"","firstName":"Camila","middleName":"Cristina Cardoso","lastName":"Gomes","suffix":""},{"id":498805782,"identity":"af7e2c36-37ce-4518-9fcc-1376a7f64754","order_by":3,"name":"Millena Arnaud Franco Igreja","email":"","orcid":"","institution":"Universidade do Estado do Pará","correspondingAuthor":false,"prefix":"","firstName":"Millena","middleName":"Arnaud Franco","lastName":"Igreja","suffix":""},{"id":498805783,"identity":"dba9599c-1c91-4eee-a625-e33c773d77eb","order_by":4,"name":"Franklyn Samudio Acosta","email":"","orcid":"","institution":"Instituto Conmemorativo Gorgas de Estudios de la Salud","correspondingAuthor":false,"prefix":"","firstName":"Franklyn","middleName":"Samudio","lastName":"Acosta","suffix":""},{"id":498805784,"identity":"1abd995e-740b-4b8a-afb2-808e2bdeac5b","order_by":5,"name":"Lourdes Maria Garcez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBACPhDBA+dWSMBYB3BqYUPVcoZkLYxtcAk8Wth7zD68qaiz6599+OHDn/Ms5Pgb2C8+5mG4k49TC88Z45lzzhxOnnEuzdhAcpuEscQBnmJjHoZnlg24tEjkGDPzth1IZjjDwyZhuE0icQMDT5rkDIbDBjhtAWv5V5csf4aH/UfiHIl6IrU0MNsZAG1hONggkWDAwH5M4gM+LTzHihnnHDucYHiGzViy4ZiE4YzDPMwGHwye4dTCz968meFNTZ293Bnmhx9/1NTJ87e3P3yQUHEHpxYYSGyAM5l5gKoJamBgsEdisz8grH4UjIJRMApGEgAALt1OH+mFhBMAAAAASUVORK5CYII=","orcid":"","institution":"Instituto Evandro Chagas/Secretaria de Vigilância em Saúde e Ambiente/Ministério da Saúde","correspondingAuthor":true,"prefix":"","firstName":"Lourdes","middleName":"Maria","lastName":"Garcez","suffix":""}],"badges":[],"createdAt":"2025-08-05 18:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7303461/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7303461/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89417969,"identity":"6bbcbf41-458f-464b-9a40-8d8ebd18b5b2","added_by":"auto","created_at":"2025-08-19 17:40:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":250564,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomic ploidy of \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147). We confirmed the presence of aneuploidy, highlighting the presence of trisomy on chromosomes 2 and 8.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7303461/v1/a6a8c5be6fa8384c8161eb67.png"},{"id":89418872,"identity":"252a2686-d72e-417d-93ba-07c1edabf8b5","added_by":"auto","created_at":"2025-08-19 17:56:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":496048,"visible":true,"origin":"","legend":"\u003cp\u003eMain functional proteins identified during the genomic annotation process of \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e. The figure depicts twenty relevant proteins with the major quantity of related genes.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7303461/v1/74287812e10619e40d096981.png"},{"id":89417970,"identity":"f30e500f-015c-4799-84b9-f0897670c365","added_by":"auto","created_at":"2025-08-19 17:40:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":280043,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of shared genes among \u003cem\u003eLeishmania\u003c/em\u003e(\u003cem\u003eLeishmania\u003c/em\u003e) \u003cem\u003emajor\u003c/em\u003e, \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003ee \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e) \u003cem\u003eguyanensis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7303461/v1/7b59c0a2438c4aeace2a9f32.png"},{"id":89418306,"identity":"6f0cb09e-d8c3-4e1b-8bae-b6ca87e760f8","added_by":"auto","created_at":"2025-08-19 17:48:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":640980,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree inferred using the \u003cem\u003ePolA1\u003c/em\u003e gene showing the \u003cem\u003eLeishmania\u003c/em\u003e clusters denoted by the clinical-epidemiological relevant categories of subgenera, complex and species.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7303461/v1/a460663a364e4c39e29fac2a.png"},{"id":93732545,"identity":"0d39d155-1fc1-4a9a-931d-08cd775c2c4b","added_by":"auto","created_at":"2025-10-17 02:32:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2910114,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7303461/v1/c6468273-b640-4312-819b-325a7430daf4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Resequencing and de novo Assembly of Leishmania (Viannia) guyanensis from Amazon region: Genome assessment, Aneuploidies, and Phylogenetic insights","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe \u003cem\u003eLeishmania\u003c/em\u003e genus (Kinetoplastida; Trypanosomatidae) includes species responsible for a spectrum of tropical zooanthroponotic diseases known as leishmaniasis. The common clinical manifestations of the disease are cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (ML), and visceral leishmaniasis (VL). The protozoans of this genus are transmitted to mammal reservoirs, including humans, by the bite of several phlebotomous species (Psychodidade: Phlebotominae), which are broadly spread in forest areas\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe diverse species of the \u003cem\u003eLeishmania\u003c/em\u003e genus cluster in four subgenera: \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e), \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eLeishmania\u003c/em\u003e), \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eSauroleishmania\u003c/em\u003e), and \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eMundinia\u003c/em\u003e). The first subgenus includes \u003cem\u003eLeishmania\u003c/em\u003e pathogens broadly distributed in the Americas. Nearly fifty \u003cem\u003eLeishmania\u003c/em\u003e species are currently known; twenty are human pathogens, including 15 endemics in the Americas\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e) subgenus is implicated in most CL and ML cases reported in Latin America\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Consequently, it is mandatory to carry out studies on the comparative genomics of the species housed in this subgenus that may reveal differences useful to explain how biological traits of \u003cem\u003eLeishmania Viannia\u003c/em\u003e parasites impact medically relevant characteristics such as drug resistance, clinical outcome, virulence, and pathogenicity.\u003c/p\u003e\u003cp\u003eFormerly, CL and ML complications were commonly associated with \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003ebraziliensis\u003c/em\u003e, the most disseminated species in the Americas. However, it is currently known that \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e causes not only single skin lesions (or multiple lesions at determined frequency) but also nasopharyngeal mucosa destruction in more severe cases\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This species occurs in several Latin American countries such as Panama, Brazil, Bolivia, Colombia, French Guiana, Suriname, Venezuela, Ecuador, and Argentina\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e transmission is strongly related to human displacement through heavy vegetation where the presence of mammal species such as \u003cem\u003eCholeopus didactylus\u003c/em\u003e, \u003cem\u003eTamandua tetradactyla\u003c/em\u003e, and \u003cem\u003eDidelphis marsupialis\u003c/em\u003e, natural reservoirs of this parasite, occur. In Brazil, the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e distribution is especially broad in the Amazon region, where drug failure rates are high, and a different treatment choice is mandatory due to the predominance of this \u003cem\u003eLeishmania\u003c/em\u003e species\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUnder pressure, \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e develops adaptative mechanisms capable of inducing drug uptake/efflux modifications in the first-line therapy used to treat CL, which causes treatment failure\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These and other biological characteristics, including virulence, pathogenicity, proliferative capacity, and immune modulation, are directly related to pathogens' genomes\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGenomic plasticity is a common feature of the \u003cem\u003eLeishmania\u003c/em\u003e genus, including aneuploidy and chromosomal fission/fusion events. Moreover, the kinetoplastid genome organization shows genes grouped in polycistronic transcription units and an evident scarcity of introns in their genes. This fact evidences a basal genomic structure without most of the more complex modular genetic elements found in other eukaryotes\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e genome has an average size of 32 Mb containing nearly 8000 protein-coding genes and 35 chromosomes due to the fusion of chromosomes 20 and 34\u003csup\u003e16\u003c/sup\u003e. When thoroughly described, this genomic data represents a source of accurate and robust information necessary for understanding parasite adaptability and survival strategies that might contribute to developing efficient therapeutic interventions.\u003c/p\u003e\u003cp\u003eIn the middle of several initiatives aimed at sequencing genomes of parasites from the \u003cem\u003eLeishmania\u003c/em\u003e genus\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, this study describes the genome of a \u003cem\u003eL\u003c/em\u003e. (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e strain from Amazonia, a \u003cem\u003eLeishmania\u003c/em\u003e species poorly investigated\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Our study is intended to elucidate not only the genetic architecture and the phylogenetic status of \u003cem\u003eL\u003c/em\u003e. (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e but also to offer further insights into the genomics of this parasite that represent a baseline to improve the diagnostic and treatment of CL and ML in the Americas.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cb\u003eEthics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study does not involve human subjects or tissue samples but only \u003cem\u003eLeishmania\u003c/em\u003e promastigotes and computational analyses, therefore ethical approval is not required.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA extraction and sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eLeishmania guyanensis\u003c/em\u003e strain was provided by a \u003cem\u003eLeishmania\u003c/em\u003e Collection (CLIOC) that is part of the Network of Biological Resource Centers for Conformity Assessment of Biological Material, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. The genomic DNA of the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e strain (MHOM/BR/75/M4147) was isolated using the Wizard\u0026reg;ฏ Genomic DNA Purification kit (Promega) following the manufacturer's instructions, rehydrating the whole DNA in a final volume of 50 \u0026micro;L. The genomic library of this parasite was then prepared using the Nextera XT DNA library preparation kit (Illumina, Inc.) following the manufacturer's protocols to subsequently be sequenced by the NexSeq 500 (Illumina, Inc.) platform using the NextSeq 500/550 High Output Kit v2.5 (300 cycles) and a pared-end sequencing approach.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReads Quality assessment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo get rid of adapters and low-quality reads (Phred\u0026thinsp;\u0026lt;\u0026thinsp;20) after the sequencing process, we used the Fastp software\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and the software FastQC\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e to depict read-quality data.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenome Assembly\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe used a \u003cem\u003ede novo\u003c/em\u003e assembly approach employing no reference genome to assemble all reads in contigs using the MEGAHIT v1.2.9 assembly software\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To create scaffolds from the assembled contigs, we used the software SSPACE v.3.0\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTaxonomical Classification of Reads and Contigs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo perform the taxonomical classification of reads and contigs obtained, we first infer the sequence homology of these sequences by a local alignment with sequences from a local curate \u003cem\u003eLeishmania\u003c/em\u003e DNA database using the Blastn algorithm\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. After sequence homology determination, we inferred the taxonomical status of reads and contigs by performing a classification based on k-mer and adding taxonomical tags to each sequence using Kraken2 software\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This classification allowed us to extract all contigs belonging to \u003cem\u003eLeishmania\u003c/em\u003e to perform the genomic annotation of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) subsequently.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenome Annotation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe genomic annotation of this parasite was performed using the AUGUSTUS software v.3.5, using as a reference the genomic structure of \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eSauroleishmania\u003c/em\u003e) \u003cem\u003etarentolae.\u003c/em\u003e We constructed a local genomic database of 69 \u003cem\u003eLeishmania\u003c/em\u003e genomes (Supplementary Table\u0026nbsp;1) retrieved from GenBank to compare genomic annotations and analyze and correct open reading frames (ORFs) positions and structures. In this way, we provided more accurate evidence of gene function during the gene annotation of this parasite. To depict the genome and manually curate ORFs obtained during the annotation process, we used the bioinformatics suite Geneious v.8.1.4\u003csup\u003e28\u003c/sup\u003e. The manual curation was finished when we analyzed the base quality and integrity of each ORFs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene Orthologs Assessment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe genomic data of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) was used to perform a Pangenome analysis aimed at identifying orthologs genes comparing this genome to the genome assembly of \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eshawi\u003c/em\u003e (GCA_962240455), \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e (GFC_000755165.1) and \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eL.\u003c/em\u003e) \u003cem\u003emajor\u003c/em\u003e (GCF_000002725.2). To do so, we used the software BLASTp v2.5.0\u003csup\u003e29\u003c/sup\u003e, Get_Homologues \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and the statistic software R using the Venn library\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMapping to reference genome, Genome Coverage, and Variant Call\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mapping to the reference genome of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e was performed using Bowtie2\u003csup\u003e32\u003c/sup\u003e, allowing an assessment of chromosomic coverage. This read mapping also allowed us to determine chromosome ploidy (chromosome copy number for each genome) by dividing each chromosome coverage by half of each thirty-five chromosome's average coverage and assuming a diploid organism. Single nucleotide polymorphisms (SNPs) and insertion/deletion (indels) events were identified using BCFtools v1.2\u003csup\u003e33\u003c/sup\u003e to detect nucleotide differences between the resequencing strain of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) and the reference genome of this species (GenBank CP103914-CP103949).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic inference\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe DNA polymerase alpha catalytic subunit (polA1) was selected as the best molecular target to infer Leishmania phylogeny after evaluating its phylogenetical signal with the TREE-PUZZEL v.5.3 algorithm\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e using the likelihood-mapping method. To assess the phylogenetic signal, we used 69 \u003cem\u003eLeishmania\u003c/em\u003e genomes (Supplementary Table\u0026nbsp;1). The polA1 gene from these genomes was identified and extracted using the software Geneious v8.1.4 to perform a phylogenetic analysis using the maximum likelihood method (ML) included in the IQTREE2 software\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e after selecting the best nucleotide substitution model by the algorithm of the software. The software parameters were set up to find the more likely tree topology testing each clade's robustness by bootstrap analysis. The choice of the best phylogenetic tree considered the genetic identity threshold among \u003cem\u003eLeishmania\u003c/em\u003e species inferred by a sequence identity matrix constructed by the software. All software parameters were set to standard conditions unless another configuration was required.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eGenome Assembly\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe genome sequencing of the \u003cem\u003eL. (V.) guyanensis\u003c/em\u003e (MHOM/BR/75/M4147) produced 9.847.950 reads with an average size of 151 pb. After filtering low-quality reads and excluding adapters and artifacts from the sequencing process, we obtained 8.839.174 high-quality reads, a reduction of 10%. The \u003cem\u003ede novo\u003c/em\u003e assembly produced 14.097 contigs with an N50 value of 4.743 bp, an average contig size of 2.239 bp, and an average genome coverage of 40.85x. The number of scaffolds constructed was identical to the number of contigs (14.097). The genome size of the \u003cem\u003eL. (V.) guyanensis\u003c/em\u003e was 31.565.639 pb (31 Mb) with a G\u0026thinsp;+\u0026thinsp;C content of 57.44%. Consequently, we obtained a new complete genome assembly (whole genome shotgun) for the \u003cem\u003eLeishmania\u003c/em\u003e isolate analyzed herein. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e gathers all the genome assembling results obtained in this study and the ones produced in earlier \u003cem\u003eLeishmania\u003c/em\u003e genomic studies. The genome assembly for \u003cem\u003eL. (V.) guyanensis\u003c/em\u003e (MHOM/BR/75/M4147) was deposited in the GenBank database under the accession number GCA_051201275.1. The data is publicly available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome/GCA_051201275.1/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_051201275.1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssembled Genomes of \u003cem\u003eLeishmania\u003c/em\u003e spp.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e\u003cp\u003eAssembled genomes of \u003cem\u003eLeishmania\u003c/em\u003e spp\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eSpecie\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eL. guyanensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eL. guyanensis\u003c/em\u003e (M4147)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eL. guyanensis\u003c/em\u003e (LgCL085)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eL. naiffi\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eL. panamensis\u003c/em\u003e (PSC-1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eL. braziliensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eL. infantum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eL. major\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(M4147)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(LnCL233)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(M2904)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(JPCM5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eFriedlin Strain\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eContigs\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14,097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10,308\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14,682\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eN50 contigs (Kb)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4743\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eScaffolds\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14,097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2,8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6,53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eN50 scaffold (Kb)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4743\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e95.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e674\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNumber of Gaps\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,557\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3,853\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e553\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e876\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e440\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAverage Coverage\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e40.85x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e30x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGenome Size (Mb)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e30.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e31.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e31.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e32.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eG\u0026thinsp;+\u0026thinsp;C content (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e57,44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e57.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e57.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e59.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e59.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eProtein-coding Genes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8,23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8,104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7,748\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8,175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8,199\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e8,272\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal Number of Genes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7698\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eN/A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8,376\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8,262\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7,933\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8,37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8,26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e8,341\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eReference\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCurrent study\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eGenbank\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCoughlan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCoughlan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLlanes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLlanes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLlanes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eIvens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eet al.\u003c/em\u003e (2018)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eet al.\u003c/em\u003e (2018)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eet al\u003c/em\u003e. (2015)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eet al\u003c/em\u003e. (2015)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eet al\u003c/em\u003e. (2015)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eet al\u003c/em\u003e. (2005)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTaxonomical Assessment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMost of the contigs produced in this study (99.95%, 14.090 contigs) were found to be related to the \u003cem\u003eLeishmania\u003c/em\u003e genus. All selected contigs showed an average nucleotide identity value of 96.29% with \u003cem\u003eLeishmania spp\u003c/em\u003e. The contigs tagged as belonging to \u003cem\u003eLeishmania\u003c/em\u003e spp. were then used in the genomic annotation process.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVariants Call and Chromosome Coverage\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe searched for variants and identified 36.665 SNPs distributed through the 35 \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e chromosomes and 8.210 \u003cem\u003eindels\u003c/em\u003e (Supplementary Table\u0026nbsp;2). R\u003cem\u003eeads\u003c/em\u003e mapping to the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e reference genome (GenBank CP103914-CP103949) identified chromosomes with coverage values between 41x and 111x (Supplementary Table\u0026nbsp;2). Additionally, there was great heterogeneity in the chromosome ploidy (values between 1.2 and 3.4), highlighting chromosomes 2 and 8 that presented a condition close to trisomy. The ploidy variations throughout \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e chromosomes are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenomic Annotation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe gene annotation of the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) revealed a lack of introns and allowed us to identify 7.698 genes, 99.9% (7.192) of which were associated with known molecular functions. After analyzing all the annotated data, we identified 3.119 different proteins related to the metabolic pathways of the parasite. The proteins with more associated genes were Calpain-like cysteine peptidase (155), protein kinase domain-containing protein (114), and transmembrane protein (75). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicts the main 20 relevant proteins identified during the annotation process.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene Orthologs Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe gene annotation of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) allowed us to study the Pangenome, orthologs genes, and \u003cem\u003ecore\u003c/em\u003e genome that altogether represent the entire group of annotated genes extant in a particular taxonomic group (MEDINI \u003cem\u003eet al\u003c/em\u003e., 2020). Therefore, we identified 6.371 genes shared by \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e, \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e, \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eshawi\u003c/em\u003e, and \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eL.\u003c/em\u003e) \u003cem\u003emajor\u003c/em\u003e. Fourteen of these genes were exclusive of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e, 13 of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e, 23 of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eshawi\u003c/em\u003e, and 299 of \u003cem\u003eL\u003c/em\u003e. (\u003cem\u003eL.\u003c/em\u003e) \u003cem\u003emajor\u003c/em\u003e. The Pangenome of these four \u003cem\u003eLeishmania\u003c/em\u003e species encompasses 7.791 genes. Regarding the \u003cem\u003eLeishmania\u003c/em\u003e species core genome, each species showed a characteristic genetic expansion (expansion in orthologs\u0026rsquo; copy numbers). For example, we found 22 expanded genes in \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e MHOM/BR/75/M4147 (Supplementary material 3). The Pangenome analysis result is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic Inference\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs a result of the phylogenetic analysis, the four \u003cem\u003eLeishmania\u003c/em\u003e subgenera were grouped into independent monophyletic clusters supported by bootstrap values higher than 90%. Furthermore, all \u003cem\u003eLeishmania\u003c/em\u003e complexes within each subgenus clustered into specific subclades supported by bootstrap values around 90% or higher. Regarding the sequence identity analyses, the species from the subgenera \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eLeishmania\u003c/em\u003e), \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eSauroleishmania\u003c/em\u003e), \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e), and \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eMundinia\u003c/em\u003e) showed sequence identity values of 90%, 99.8%, 96.8%, and 84.7%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe genome assembling of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) performed herein strengthens the extant genomic data of this Leishmania species, providing excellent values of completeness and coherence when compared with the current \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e reference genome. An N50 value of 6.029 bp supported the good quality of the genome assembly as the parameter suggested that most of the contigs were long enough and well-assembled. The clear evidence that the scaffold number obtained was identical to the contigs number also supports the completeness of the assembled contigs, which are long enough and need no additional connections as links or bridges to create scaffolds. This fact reflects the good quality of the genome assembly obtained by the \u003cem\u003ede novo\u003c/em\u003e method. Certainly, the quality filtering and taxonomical identification of the reads were critical for obtaining the correct genome structure since 9% of the reads were classified as sequencing artifacts.\u003c/p\u003e\u003cp\u003eThe reference genome of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (GenBank CP103914-CP103949) presented an average coverage value of 27.0x, while the coverage value obtained in this study for the same \u003cem\u003eLeishmania\u003c/em\u003e strain was 40.85x. This higher coverage might have helped to acquire more accurate results in other genome-based analyses, such as SNPs identification and variant calls. Moreover, this study includes a robust genome annotation for this \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e strain, information that is still missing in genome databases such as TriTrypDB and GenBank.\u003c/p\u003e\u003cp\u003eAs the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e genome assembly performed in this study, the assembly reported earlier for the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e LgCL085 genome\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e presented good assembling parameters. However, a different bioinformatic workflow including assembly corrections, gap filling, and junction joining was necessary to complete the genome assembly. Both \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (LgCL085) and \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e (PSC-1) genomes available in the GenBank were characterized via \u003cem\u003ede novo\u003c/em\u003e assembly\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, representing the only strains from the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e complex with complete genomes available in genome databases. The genomic characterization of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) performed in this study complements the genomic data of this species, providing indicative parameters of quality and completeness lacking in most genomic studies of \u003cem\u003eLeishmania\u003c/em\u003e. Generally, the genome assembly of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) obtained herein showed similar genomic characteristics as the ones previously reported for \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003ebraziliensis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eLeishmania\u003c/em\u003e) \u003cem\u003eamazonensis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, which present genome sizes of nearly 30Mb, average G\u0026thinsp;+\u0026thinsp;C content of 57%, and a value of predicted gene quantity close to 8000. This result indicates convergency among methods used to assemble \u003cem\u003eLeishmania\u003c/em\u003e genomes, highlighting the importance of resequencing previously characterized \u003cem\u003eLeishmania\u003c/em\u003e species, especially those with intrinsic epidemiological and clinical relevancy.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e genome obtained in this study presented chromosome number variation, a biological event known as aneuploidy\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Aneuploidy tolerance is a trypanosomatid ancestral characteristic that might be prevalent in a monophyletic clade that underwent no significant genomic reorganization\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This characteristic frequently occurs in the \u003cem\u003eLeishmania\u003c/em\u003e genus without defined patterns among species, resulting in karyotype diversity within the parasite lineages (mosaic aneuploidy)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The present analysis reveals that the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e karyotype varies depending on the genomic reference used for gene mapping. We found that when the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e genome is mapped to the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003ebraziliensis\u003c/em\u003e genome, chromosomes 8 and 31 seem to be supernumerary. Nonetheless, when the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e genome is mapped to the reference genome for this species, the karyotype reveals a tendency to the disomic state, with chromosomes 1, 2, 3, 8, 31, 33, and 34 showing coverage values higher than 2.5. the intermediary values observed in the chromosomic coverage assessment might result from a mixed cellular population where chromosomic ploidy variation exists in the same sample, a common occurrence in \u003cem\u003eLeishmania\u003c/em\u003e spp.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eA more significant deviation from the disomic state was observed during the read mapping in chromosomes 2 and 8. This fact also supports that the genomic reference choice influence significantly affects karyotype even among species belonging to the same subgenus, highlighting the importance of using a specific reference genome from the same species to perform more accurate analyses. Even though \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) was mapped to the reference genome of this species, it was found 36,665 SNPs in this \u003cem\u003eLeishmania\u003c/em\u003e strain. This genetic variability might arise during parasite culture in different microenvironments, resulting in a genetic adaptation favoring parasite survival to various environmental stimuli\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe study carried out herein complements the genetic architecture previously described for \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) (reference) identifying genes (7726) and describing functional proteins. In this sense, the gene ontology analyses of the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (LgCL085) revealed the presence of 8230 protein-coding genes. Most of these proteins were associated with ortholog genes reported in the \u003cem\u003eViannia\u003c/em\u003e subgenus whose copy numbers vary from one species to another\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Due to the polycistronic nature of this genus, each protein is not strictly associated with one gene, and there are also no coding DNA and ORFs coding for more than one protein. This fact was verified after finding a high level of genes associated with one functional protein. The initial description of 3128 protein-coding genes in \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e might aid in disclosing the translational composition of this species, a fact of paramount importance for understanding its cellular and metabolic processes\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe gene annotation was of value to identify essential elements including start and stop codons, exon-intron junctions, protein-coding regions, and promoters and regulatory regions. As a result, we observed a lack of introns in most of the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e protein-coding genes, evidence previously reported in genomic studies of \u003cem\u003eLeishmania\u003c/em\u003e earlier\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, and that could be linked to inherent polycistronic characteristics of the \u003cem\u003eLeishmania\u003c/em\u003e genus. This polycistronic region includes a DNA region processed during transcription to produce a long polycistronic RNA including non-related genes organized into a genomic architecture resembling the prokaryotes' gene organization\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe workflow described herein identifies proteins and ortholog genes in microorganisms in general and has also been used earlier to assess \u003cem\u003eLeishmania\u003c/em\u003e spp. genomes\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Using this strategy, we identified ortholog genes shared by \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e, \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e, and \u003cem\u003eL\u003c/em\u003e. (\u003cem\u003eL.\u003c/em\u003e) \u003cem\u003emajor\u003c/em\u003e and a reduced group of genes (48) restricted to \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e. Among these three species, \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eL.\u003c/em\u003e) \u003cem\u003emajor\u003c/em\u003e showed the highest number of genes unrelated to the other species (1631), probably because it belongs to a different \u003cem\u003eLeishmania\u003c/em\u003e subgenus. Conversely, we found fewer exclusive genes between \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e and \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e. Generally, gene ortholog studies have indicated low quantities of species-specific genes in \u003cem\u003eLeishmania\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Additionally, we observed a trend indicating that closely related \u003cem\u003eLeishmania\u003c/em\u003e species from the same complex share more ortholog genes in the Pangenome than those \u003cem\u003eLeishmania\u003c/em\u003e parasites from different subgenus. This might be explained by the great divergency bridge between \u003cem\u003eLeishmania\u003c/em\u003e spp. from different subgenus due to the parasites' adaptation to conditions found in each transmission cycle. This biological adjustment of parasites from different \u003cem\u003eLeishmania\u003c/em\u003e subgenera during the evolutionary process might explain the functional diversity found in the \u003cem\u003eLeishmania\u003c/em\u003e genus nowadays. Indeed, the functional diversity produced in this way might account for the differential group of orthologs belonging to the different taxonomic units within the \u003cem\u003eLeishmania spp\u003c/em\u003e. Pangenome.\u003c/p\u003e\u003cp\u003eThe phylogenetic analysis was important for validating the genome assembly data of \u003cem\u003eL\u003c/em\u003e. (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e (MHOM/BR/75/M4147) because it might explain any discrepancies found during the assembly process\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The analysis using TREE-PUZZLE software showed a high phylogenetic signal of the \u003cem\u003epolA1\u003c/em\u003e gene favoring the construction of a well-supported phylogenetic tree. This molecular target has been used to identify \u003cem\u003eLeishmania\u003c/em\u003e species\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, showing to be a potential candidate to differentiate cryptic species such as \u003cem\u003eL\u003c/em\u003e. (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e and \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e, \u003cem\u003eLeishmania\u003c/em\u003e species associated with tegumentary leishmaniasis in the Americas. This fact is important because, in the last 24 years, the validation of \u003cem\u003eLeishmania\u003c/em\u003e species has been a matter of discussion. The main taxonomical problem lies in the initial \u003cem\u003eLeishmania\u003c/em\u003e taxonomical proposal based mainly on the biological and eco-epidemiological characteristics of the current species\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe molecular marker \u003cem\u003epolA1\u003c/em\u003e was used earlier to distinguish and group \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eMundinia\u003c/em\u003e) \u003cem\u003emartiniquensis\u003c/em\u003e by phylogenetics when the Multilocus Enzyme Electrophorese analyses (MEE) could not differentiate this \u003cem\u003eLeishmania\u003c/em\u003e species\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In this study, the phylogenetic analysis using the \u003cem\u003epolA1\u003c/em\u003e gene clustered in different clades of cryptic species, some \u003cem\u003eLeishmania\u003c/em\u003e species from the same complex, such as \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eLeishmania\u003c/em\u003e) \u003cem\u003edonovani\u003c/em\u003e and \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eL.\u003c/em\u003e) \u003cem\u003einfantum; L.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e and \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e; \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003ebraziliensis\u003c/em\u003e and \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e) \u003cem\u003eperuviana.\u003c/em\u003e All sequences from \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e and \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e species clustered into different monophyletic clades, unlike studies based on an HSP70-based approach that presented problems differentiating these cryptic species\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Phylogeny based on the HSP70 gene in \u003cem\u003eLeishmania\u003c/em\u003e has been of help with the subgenera assignment and raised some important questions on the validity of some species belonging to the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e complex\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The phylogenetic analysis performed herein showed the potential of the \u003cem\u003epolA1\u003c/em\u003e gene for the \u003cem\u003eLeishmania\u003c/em\u003e complexes validation when compared with the \u003cem\u003eLeishmania\u003c/em\u003e HSP70 gene region. The main pitfall reported in HSP70-based protocols is their problems discerning some species from the \u003cem\u003eViannia\u003c/em\u003e subgenus since the geographical distribution of the \u003cem\u003eLeishmania\u003c/em\u003e species in the Americas has favored a high ancestral genetic flux\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEven using the Multilocus Sequence Typing approach (MLST), the phylogenetic differentiation between \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e and \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003epanamensis\u003c/em\u003e from the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e complex is still problematic\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The advances in the etiological characterization of the \u003cem\u003eLeishmania\u003c/em\u003e genus have suggested modifications in the taxonomic nomenclature of species\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and the validation of complexes within subgenera\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. To do so, it is preconized to use and look at DNA-sequence-based strategies instead of traditional methods based on isoenzymes, a recommendation we followed by using a molecular approach based on the \u003cem\u003ePolA1\u003c/em\u003e gene. The phylogenetic species concept is based on creating a monophyletic group with characteristics different from other groups and following a model of parental ancestry\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Consequently, the tree topology obtained herein using the \u003cem\u003epolA1\u003c/em\u003e gene from \u003cem\u003eLeishmania\u003c/em\u003e species suggests the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis\u003c/em\u003e species complex validation. Furthermore, the phylogenetic tree indicated nucleotide identity grades of at least 90% among species from the same subgenus except the \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eMundinia\u003c/em\u003e) subgenus (84.7%). This parameter indicates the number of identical sites among clade components. Observing the tree topology, it is evident that \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eM.\u003c/em\u003e) \u003cem\u003emartiniquensis\u003c/em\u003e showed the highest phylogenetic distance when compared visually with the rest of the species from the \u003cem\u003eMundinia\u003c/em\u003e subgenus, therefore, influencing the global nucleotide identity value assessed for this group. Indeed, when removing the \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003emartiniquensis\u003c/em\u003e from the analysis, the \u003cem\u003eMundinia\u003c/em\u003e clade clustered species with a nucleotide identity value of 95%, an increasing rate of more than 10%. This result suggests that \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003emartiniquensis\u003c/em\u003e might be the most divergent species of the whole \u003cem\u003eLeishmania\u003c/em\u003e genus.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe genomic assembly performed in this study showed equivalent results to the rest of the \u003cem\u003eLeishmania\u003c/em\u003e assembly data obtained earlier complementing and improving the genomic architecture data of \u003cem\u003eL.\u003c/em\u003e (\u003cem\u003eV.\u003c/em\u003e) \u003cem\u003eguyanensis.\u003c/em\u003e Further analysis such as variant calls, aneuploidy studies, and the genomic annotation performed herein add important information to the initial genomic description of this \u003cem\u003eLeishmania\u003c/em\u003e species and describe relevant functional proteins important for \u003cem\u003eLeishmania\u003c/em\u003e etiopathogenesis. The polyA1 marker proved to be a promising candidate for developing phylogenetic studies of \u003cem\u003eLeishmania\u003c/em\u003e easily identifying \u003cem\u003eLeishmania\u003c/em\u003e subgenera and complexes and showing outstanding discrimination power to validate and differentiate \u003cem\u003eLeishmania\u003c/em\u003e species belonging to these two taxonomical categories. The phylogenetic analysis also allowed us to identify a high divergency level within the \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eMundinia\u003c/em\u003e) subgenus, highlighting \u003cem\u003eL\u003c/em\u003e. (\u003cem\u003eM\u003c/em\u003e.) \u003cem\u003emartiniquensis\u003c/em\u003e as probably the most divergent species in the \u003cem\u003eLeishmania\u003c/em\u003e genus. Overall, this study validates the efficiency of modern methods based on sequencing, genomic analysis, and phylogeny in the \u003cem\u003eLeishmania\u003c/em\u003e species characterization process. Lastly, this efficiency obtained by up-to-date methods facilitates understanding important molecular, biological, and epidemiological aspects of \u003cem\u003eLeishmania spp.\u003c/em\u003e in the Americas paving the way for further valuable studies on leishmaniasis in this continent.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the following organizations in Brazil and Panama for supporting researchers and students involved in this study: \u003cem\u003ePrograma de P\u0026oacute;s-Gradua\u0026ccedil;\u0026atilde;o Bionorte - Rede de Biodiversidade e Biotecnologia da Amaz\u0026ocirc;nia, Universidade Federal do Par\u0026aacute;\u003c/em\u003e, Brazil; \u003cem\u003ePrograma Institucional de Bolsas de Inicia\u0026ccedil;\u0026atilde;o Cient\u0026iacute;fica (Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico \u0026ndash; CNPq), Instituto Evandro Chagas\u003c/em\u003e, Brazil; \u003cem\u003eInstituto Conmemorativo Gorgas de Estudios de la Salud\u003c/em\u003e, Panama and \u003cem\u003eSistema Nacional de Investigaci\u0026oacute;n\u003c/em\u003e, Panama.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL.G.A.C.\u003c/strong\u003e wrote the initial manuscript draft, made formal analysis and generated figures. \u003cstrong\u003eE.C.S.J.\u003c/strong\u003e devised the computational strategy, did formal analysis and results interpretation, reviewed and edited the draft concerning bioinformatic aspects. \u003cstrong\u003eC.C.C.G.\u003c/strong\u003e analyzed the formal chromosomal ploidy, wrote respective results and formatted references. \u003cstrong\u003eM.A.F.I.\u003c/strong\u003e improved the formal genome assembling workflow and composed the table; \u003cstrong\u003eF.S.A.\u003c/strong\u003e reviewed the results analysis and interpretation; wrote and translated the final draft fitting to the journal format. \u003cstrong\u003eL.M.G.\u003c/strong\u003e Contributed to the conception, design and planning of the study, fundraising, results analysis and interpretation, final draft review and editing, supervised the research team and mentored all contributing students. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancier of studies and projects (FINEP), Brazil, agreement number 01.22.0495.00; Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil, within the scope of Notice n\u003csup\u003eo\u003c/sup\u003e. 13/2020 - Postgraduate Development Program - Process: 88887.919409/2023-00.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in the GenBank database under the accession number GCA_051201275.1. The data is publicly available at https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_051201275.1/.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and request for materials should be addressed to Lourdes Garcez\u003c/p\u003e\n\u003cp\u003eReprints and permissions information is available at www.nature.com/reprints.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral about jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRights and permissions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOpen Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlencar, R. B., Justiniano, S. C. B. \u0026amp; Scarpassa, V. M. Morphological Description of the Immature Stages of Nyssomyia umbratilis (Ward \u0026amp; Frahia) (Diptera: Psychodidae: Phlebotominae), the Main Vector of \u003cem\u003eLeishmania guyanensis\u003c/em\u003e Floch (Kinetoplastida: Trypanosomatidae) in the Brazilian Amazon Region. \u003cem\u003eNeotrop. 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C. \u0026amp; Porrozzi, R. Considera\u0026ccedil;\u0026otilde;es sobre taxonomia do g\u0026ecirc;nero \u003cem\u003eLeishmania\u003c/em\u003e. In: CONCEI\u0026Ccedil;\u0026Atilde;O-SILVA, F.; ALVES, C. R. Leishmanioses do Continente Americano. 1\u0026ordf; ed. Rio de Janeiro (ed. Editora Fiocruz) 39\u0026ndash;51, (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Leishmania guyanensis, genome assembly, genomic annotation, polA1, phylogeny","lastPublishedDoi":"10.21203/rs.3.rs-7303461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7303461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe \u003cem\u003eLeishmania\u003c/em\u003e genus includes 20 human pathogenic species, 15 occurring in the Americas. In Amazonia, \u003cem\u003eLeishmania guyanensis\u003c/em\u003e is frequently associated with therapeutic failure, what is related to genetic plasticity and adaptability during drug exposure. The genomic studies of this species are important to understand different aspects of parasite biology including genetic adaptability to different environments and possible therapeutic alternatives. Taking this into account, we sequenced a \u003cem\u003eL. guyanensis\u003c/em\u003e strain (MHOM/BR/75/M4147), performed the quality assessment of the reads (FASTP), and the genome assembly (MEGAHIT). The taxonomic classification was accomplished using BLASTn and Kraken2 software, and the annotation of \u003cem\u003eLeishmania\u003c/em\u003e contigs using Augustus v.3.5. The assembled genome showed a size of 31 Mb, N50\u0026thinsp;=\u0026thinsp;4.743 bp, genome coverage of 40.85x, and 99,5% of its contigs were related to \u003cem\u003eLeishmania\u003c/em\u003e. We confirmed the presence of 36,665 SNPs, 8210 Indels, and aneuploidies. After annotation, we identified 3119 proteins associated with well-known molecular functions in \u003cem\u003eL. guyanensis\u003c/em\u003e and 6.371 ortholog genes shared by \u003cem\u003eL. guyanensis\u003c/em\u003e, \u003cem\u003eL. major\u003c/em\u003e, and \u003cem\u003eL. panamensis\u003c/em\u003e. The phylogenetic analysis using the \u003cem\u003epolA1\u003c/em\u003e gene correctly grouped \u003cem\u003eL. guyanensis\u003c/em\u003e showing discriminatory potential to identify all \u003cem\u003eLeishmania\u003c/em\u003e species, highlighting \u003cem\u003eL. martiniquensis\u003c/em\u003e as the more divergent species within the \u003cem\u003eLeishmania\u003c/em\u003e genus. The overall results complement the extant genomic data of \u003cem\u003eL. guyanensis\u003c/em\u003e and encourage progress in the species-specific diagnostic of \u003cem\u003eLeishmania\u003c/em\u003e spp.\u003c/p\u003e","manuscriptTitle":"Resequencing and de novo Assembly of Leishmania (Viannia) guyanensis from Amazon region: Genome assessment, Aneuploidies, and Phylogenetic insights","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 17:40:07","doi":"10.21203/rs.3.rs-7303461/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7c463378-baeb-4c73-95f3-699d94d1a137","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52978681,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":52978682,"name":"Biological sciences/Evolution"},{"id":52978683,"name":"Biological sciences/Genetics"},{"id":52978684,"name":"Biological sciences/Microbiology"},{"id":52978685,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-10-17T02:24:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 17:40:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7303461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7303461","identity":"rs-7303461","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}