A complete Leishmania donovani reference genome identifies novel genetic variations associated with virulence

Patrick Lypaczewski,Johanna Hoshizaki,Amanpreet Kaur,Laura-Isobel McCall,John Torcivia-Rodriguez,Vahan Simonyan,Greg Matlashewski,Wen-Wei Zhang,Ken Dewar

doi:10.1038/s41598-018-34812-x

Abstract

Leishmania donovani is responsible for visceral leishmaniasis, a neglected and lethal parasitic disease with limited treatment options and no vaccine. The study of L. donovani has been hindered by the lack of a high-quality reference genome and this can impact experimental outcomes including the identification of virulence genes, drug targets and vaccine development. We therefore generated a complete genome assembly by deep sequencing using a combination of second generation (Illumina) and third generation (PacBio) sequencing technologies. Compared to the current L. donovani assembly, the genome assembly reported within resulted in the closure over 2,000 gaps, the extension of several chromosomes up to telomeric repeats and the re-annotation of close to 15% of protein coding genes and the annotation of hundreds of non-coding RNA genes. It was possible to correctly assemble the highly repetitive A2 and Amastin virulence gene clusters. A comparative sequence analysis using the improved reference genome confirmed 70 published and identified 15 novel genomic differences between closely related visceral and atypical cutaneous disease-causing L. donovani strains providing a more complete map of genes associated with virulence and visceral organ tropism. Bioinformatic tools including protein variation effect analyzer and basic local alignment search tool were used to prioritize a list of potential virulence genes based on mutation severity, gene conservation and function. This complete genome assembly and novel information on virulence factors will support the identification of new drug targets and the development of a vaccine for L. donovani.

Highlights

Visceral Leishmaniasis (VL) is the second most lethal parasitic disease following malaria and is prevalent throughout underdeveloped and tropical regions of the world
Due to its repetitive nature, the A2 gene cluster is misassembled in all Leishmania genomes generated using second generation sequencing, and only resolved in a recent resequencing effort targeted to L. infantum exploiting the long-read capabilities of Pacific Biosciences (PacBio) sequencing which resulted in a complete genome assembly[18]
The current L. donovani genome was obtained from second generation sequencing and no precise DNA or complete protein sequences are available for any A2 protein in L. donovani, hindering the comparison of A2 genes in visceral disease-causing strains or using mass spectrometry to identify A2 proteins which relies on accurate genome sequences for protein identification

Summary

Introduction

Visceral Leishmaniasis (VL) is the second most lethal parasitic disease following malaria and is prevalent throughout underdeveloped and tropical regions of the world. Long read sequencing or “third-generation” sequencing refers to more recent technologies including Oxford NanoPore[12] and Pacific Biosciences (PacBio)[13] that can result in reads ranging up to 50 kb or 100 kb that are capable of generating more complete genomic assemblies, provided the read lengths traverse across repetitive elements One such highly repetitive cluster is the A2 gene family from L. donovani considered to be an important virulence factor and is necessary for survival in visceral organs[14,15] and protection against host response stress[16,17]. We have combined second and third generation sequencing to generate a complete assembly of the L. donovani genome from the strain responsible for cutaneous leishmaniasis (CL) in Sri Lanka[4,19] This new assembly enabled the generation of an improved genome annotation and an unbiased analysis of chromosome synteny comparing L. donovani and L. major genes and strand switch transcription units. This study further enabled re-annotation of much of the genome highlighting the importance of a complete reference assembly to support future functional genomic and proteomic studies involving the L. donovani pathogen

Methods

Results

Conclusion