Comparative Genomics of Leishmania donovani to Uncover Pathogenicity Genes

A Kaur¹, J Wasserscheid^1,2, H Djambazian^1,2, S Ranasingha³, L McCall^1,4, W W Zhang¹, G Matlashewski¹, K Dewar^1,2

1. McGill University; 2. McGill University and Genome Quebec Innovation Centre; 3. University of Sri Jayewardenepura Gangodawila, Sri Lanka; 4. University of California San Francisco

With over 350 million cases reported annually, leishmaniasis, a disease caused by pathogens of genus Leishmania, is the second largest parasitic killer in the world. Two different strains of the same species of Leishmania viz. L. donovani have been linked to different types of leishmaniasis in Sri Lanka; an avirulent strain (SLCL) that causes cutaneous leishmaniasis and a virulent strain (SLVL) that causes visceral leishmaniasis. A laboratory-derived strain (SLCL_IV), which was initially avirulent, became virulent upon iterative infections of a mouse host. To uncover the mutations that allowed the avirulent strain to become virulent, this study has performed genome wide comparisons of the three strains. As these “strains” are all populations of individuals, we are using a population genetics approach rather than a search for novel, fixed mutations.

By combining several DNA sequencing technologies like PacBio and Illumina MiSeq a high quality genome assembly for SLCL_IV strain was constructed. The Illumina technology was then also used for genome wide resequencing of the three strains to identify SNPs associated with increased virulence in SLCL_IV.

Using MiSeq reads which are short yet highly accurate, a genome wide comparison of the three strains was performed. After alignment of the three Illumina MiSeq datasets to the SLCL_IV PacBio assembly, we tabulated coverage in perfect matches (ph; identity over 100% length) and in high-scoring, but not perfect, hits (gh; >=83% identity). By calculating the proportion of perfect hits versus high scoring hits for each sample, we were able to determine the ‘allele shifts’ in all three strains at all positions in the genome. By discriminating between perfect and high scoring, we have a generalized approach that can identify substitutions (SNPs) as well as indels and rearrangements. Our method has led to the identification of non-synonomous mutation SNPs in two genes. Knowledge of how these SNPs and genes could affect virulence in L. donovani may lead to a better understanding of pathology of leishmaniasis.