Institute of Tropical Medicine Antwerp, Molecular Parasitology Unit, Antwerp, Belgium.
Wellcome Sanger Institute, Hinxton, UK.
Sci Rep. 2020 Sep 14;10(1):15043. doi: 10.1038/s41598-020-71882-2.
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.
在这里,我们报告了一项开拓性研究,为进一步的利什曼原虫单细胞基因组学研究铺平了道路。首先,我们比较了两种市售的全基因组扩增(WGA)试剂盒,即 PicoPLEX 和 RepliG,在少量利什曼原虫 DNA 上的性能,测试它们保留特定遗传变异(包括非整倍体水平和 SNP)的能力。我们在这里表明,WGA 方法的选择应取决于计划的下游遗传分析,PicoPLEX 和 RepliG 分别更适合于非整倍体和 SNP 调用。这种比较使我们能够评估和优化相应的生物信息学方法。由于 PicoPLEX 被证明是研究单细胞非整倍体的首选方法,因此在第二步中,我们应用该方法对通过荧光激活细胞分选(FACS)分选的 L. braziliensis 单细胞进行了研究。即使在 28 个单细胞中也实现了测序深度,从而可以准确估计体细胞数。在 25 个细胞中观察到具有三个非整倍体染色体的优势核型,而在其他细胞中观察到两种不同的次要核型。因此,我们的方法能够检测到非整倍体镶嵌性,并为进一步与高通量单细胞基因组学方法相吻合提供了坚实的基础。