Genome Sequencing of Leishmania infantum Causing Cutaneous Leishmaniosis from a Turkish Isolate with Next-Generation Sequencing Technology.

Purpose: Leishmania subgenus Leishmania causes leishmaniosis, which is a chronic systemic disease in humans and animals, in which the skin and visceral organs can be affected. The disease generally consists of three different clinical types in humans: visceral (kala-azar, VL), cutaneous (CL) and mucocutaneous leishmaniosis (MCL). According to the World Health Organization (WHO), leishmaniosis is still one of the world's most neglected diseases. It has been nearly 13-14 years since the completion of the first complete genome sequence of a Leishmania parasite. However, much information about these parasites remains to be elucidated, such as the causes of differences in tissue tropism. The aim of this study is to perform the whole-genome sequencing of Leishmania infantum causing cutaneous leishmaniosis from a Turkish isolate with next-generation sequencing technology.

Methods: Genomic sequencing was performed on the Illumina HiSeq 2500 platform. The TruSeq Nano DNA Low Throughput Library Prep Kit, compatible with the Illumina HiSeq 2500 platform, was used to generate the library. Synthesis sequencing (SBS) was performed with a HiSeq Rapid SBS Kit v2 to generate single-fragment reads (2 × 150 bp; PE) with two fragment end-to-end assemblies. Bioinformatics analyses were performed on the Geneious 11.0.5. ( www.genius.com ) platform.

Results: In our study, a high-quality whole-genome sequence (WGS) of L. infantum was successfully generated, and a total of 32,009,137 base pairs of genomic DNA from 36 chromosomes were obtained. The resulting genomic DNA sequence was submitted to the US National Center for Biotechnology Information (NCBI) GenBank ( www.ncbi.nlm.nih.gov ) database and registered under the name Leishmania infantum_TR01 (Lin_TR01). The following accession numbers were assigned by NCBI to the 36 chromosomes of the Lin_TR01 genome: CP027807, CP027810, CP027808, CP027811, CP027809, CP027812, CP027813, CP027814, CP027817, CP027818, CP027819, CP027815, CP027821, CP027816, CP027823, CP027820, CP027822, CP027824, CP027825, CP027826, CP027827, CP027828, CP027829, CP027830, CP027831, CP027832, CP027833, CP027834, CP027835, CP027836, CP027837, CP027838, CP027839, CP027840, CP027841, CP027842. As a result of the annotation of the Lin_TR01 genome, 3153 polymorphisms, 8324 genes, 8199 CDSs, 8109 mRNAs, 67 tRNAs, 11 rRNAs and 58 ncRNA were identified. Among the 8199 CDS obtained, 5278 encode hypothetical proteins.

Conclusion: In this study, a high-quality WGS of Leishmania infantum was successfully obtained for the first time in Turkey. According to a review of WGS studies on this subject, the Lin_TR01 strain is the first strain to be isolated from cutaneous leishmaniosis. The reference genome of L. infantum JPCM5 (Peacock et al., 2007) was obtained from a visceral leishmaniosis case, in accordance with the classical tissue and organ tropism of the species. Lin_TR01 is the second whole-genome-sequenced strain in the world after the JPCM5 strain. The Lin_TR01 genome is the only L. infantum whole-genome sequence that is completed assembly level from 36 chromosomes among the genomes obtained thus far ( https://www.ncbi.nlm.nih.gov/genome/genomes/249 ).