Evaluation of whole genome amplification and bioinformatic methods for the characterization of Leishmania genomes at a single cell level

Abstract

Here, we report a pilot study paving the way for further single cell genomics studies in Leishmania. First, the performances of two commercially available kits for Whole Genome Amplification (WGA), PicoPLEX and RepliG were compared on small amounts of Leishmania donovani DNA, testing their ability to preserve specific genetic variations, including aneuploidy levels and SNPs. We show here that the choice of WGA method should be determined by the planned downstream genetic analysis, PicoPLEX and RepliG performing better for aneuploidy and SNP calling, respectively. This comparison allowed us to evaluate and optimize corresponding bio-informatic methods. As PicoPLEX was shown to be the preferred method for studying single cell aneuploidy, this method was applied in a second step, on single cells of L. braziliensis, which were sorted by fluorescence activated cell sorting (FACS). Even sequencing depth was achieved in 28 single cells, allowing accurate somy estimation. A dominant karyotype with three aneuploid chromosomes was observed in 25 cells, while two different minor karyotypes were observed in the other cells. Our method thus allowed the detection of aneuploidy mosaicism, and provides a solid basis which can be further refined to concur with higher-throughput single cell genomic methods.

High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani

“…Since Leishmania chromosomes are biased in GC content (Imamura et al, 2020), with small chromosomes (Chr1 to Chr5) displaying a higher GC content than others, amplification bias due to differences in GC content can have a negative impact in the determination of the copy number of these chromosomes. Plotting the distribution of NMD values leads to different peaks, each peak representing one of the somy values, however, the peaks of these small chromosomes with high GC content are shifted relative to the other chromosomes (supp.…”

Exploring the evolution and adaptive role of mosaic aneuploidy in a clonal Leishmania donovani population using high throughput single cell genome sequencing

“…The average effective coverage depth per cell was 0,14x, with only 2 cells displaying a depth coverage higher than 1x (Figure S1A). However, read mapping was evenly distributed across chromosomes in general(Figure S1B), which allows somy estimation despite low depth 18 . From 512 clusters, 452 passed the bin-to-bin variability filtering, representing a total of 1560 cells.…”

“…Accordingly, pioneer FISH-based studies should be complemented and refined by single cell genome sequencing (SCGS). In a previous study 18 , we made a first step in that direction by combining FACS-based sorting of single Leishmania cells with whole genome amplification (WGA) and whole genomic sequencing (WGS). In this pilot study, we evaluated different WGA and bioinformatic methods and detected 3 different karyotypes among 28 single cells of L. braziliensis 18 .…”

“…In a previous study 18 , we made a first step in that direction by combining FACS-based sorting of single Leishmania cells with whole genome amplification (WGA) and whole genomic sequencing (WGS). In this pilot study, we evaluated different WGA and bioinformatic methods and detected 3 different karyotypes among 28 single cells of L. braziliensis 18 . Here, we made one step beyond, applying a droplet-based platform for single cell genomics, in order to undertake the first high-throughput study of MA in Leishmania.…”

Long-term Hematopoietic Stem Cells as Sanctuary Niche during Treatment Failure in Visceral Leishmaniasis

“…Several molecules are involved in the internalization of Leishmania parasites [47], but dditional research will be needed to understand Leishmania entry into LT-HSCs. Multiomics pro ling of infected LT-HSCs and the intracellular parasites in envisaged to further elucidate the host-parasite interactions in this particular cellular niche [48].…”