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).

中文翻译：

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使用下一代测序技术对土耳其分离株引起皮肤利什曼病的婴儿利什曼原虫进行基因组测序。

目的

利什曼原虫亚属利什曼原虫会引起利什曼原虫病，这是一种人类和动物的慢性全身性疾病，其中皮肤和内脏器官会受到影响。该疾病通常由人类的三种不同临床类型组成：内脏（黑热病，VL）、皮肤（CL）和皮肤粘膜利什曼病（MCL）。据世界卫生组织（WHO）称，利什曼病仍然是世界上最被忽视的疾病之一。自从完成第一个利什曼原虫寄生虫的完整基因组序列以来，已经过去了将近 13-14 年。然而，关于这些寄生虫的许多信息仍有待阐明，例如组织趋向性差异的原因。本研究的目的是对婴儿利什曼原虫进行全基因组测序。 使用下一代测序技术从土耳其分离株中引起皮肤利什曼病。

方法

基因组测序在 Illumina HiSeq 2500 平台上进行。TruSeq Nano DNA 低通量文库制备试剂盒与 Illumina HiSeq 2500 平台兼容，用于生成文库。使用 HiSeq Rapid SBS Kit v2 进行合成测序 (SBS)，以生成具有两个片段端到端组装的单片段读数（2 × 150 bp；PE）。在 Geneious 11.0.5 上进行生物信息学分析。(www.genius.com) 平台。

结果

在我们的研究中，成功生成了婴儿乳杆菌的高质量全基因组序列（WGS），从 36 条染色体中获得了总共 32,009,137 个基因组 DNA 碱基对。得到的基因组 DNA 序列提交给美国国家生物技术信息中心 (NCBI) GenBank (www.ncbi.nlm.nih.gov) 数据库，并以Leishmania infantum的名称注册_TR01 (Lin_TR01)。下述的登录号是由NCBI分配给36条染色体的Lin_TR01基因组：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。作为 Lin_TR01 基因组注释的结果，鉴定了 3153 个多态性、8324 个基因、8199 个 CDS、8109 个 mRNA、67 个 tRNA、11 个 rRNA 和 58 个 ncRNA。在获得的 8199 个 CDS 中，5278 个编码假设的蛋白质。

结论

本研究首次在土耳其成功获得了高质量的婴儿利什曼原虫WGS 。根据对该主题的 WGS 研究的回顾，Lin_TR01 菌株是第一个从皮肤利什曼病中分离出来的菌株。L. infantum JPCM5 (Peacock et al., 2007)的参考基因组是从一个内脏利什曼病病例中获得的，符合该物种的经典组织和器官趋向性。Lin_TR01 是继 JPCM5 菌株之后世界上第二个全基因组测序菌株。Lin_TR01 基因组是迄今为止获得的基因组中唯一从 36 条染色体完成组装的婴儿乳杆菌全基因组序列 (https://www.ncbi.nlm.nih.gov/genome/genomes/249)。