Objective: To detect somatic DNA copy number variants (CNVs) in dopaminergic neurons isolated from human substantia nigra, using shallow whole genome sequencing (WGS).

Background: Somatic (post-zygotic) mutations lead to mosaicism (genomic diversity within an individual), and may have a role in neurodegeneration [1]. Technical advances have allowed sequencing the genome of single cells, which can be performed at very low coverage for CNV detection. We recently reported somatic CNVs of alpha-synuclein (SNCA) in substantia nigra in a targeted study, commoner in dopaminergic neurons of synucleinopathies than controls, with possible clinical correlates [2].

Method: Material: A human control substantia nigra was used. Cell / nuclear isolation: (1) Laser-capture microdissection of cells identified by neuromelanin. (2) Nuclear fraction isolation from tissue, with dopaminergic nuclei labelled by an appropriate antibody. We selected nuclei either by fluorescence-activated nuclear sorting (FANS), or a manual device on an inverted microscope (Qiascout). Whole genome amplification (WGA), sequencing, and analysis: Four kits were compared (GenomePlex, TruePrime, RepliG, Picoplex). Libraries were sequenced on Illumina MiSeq, aiming to obtain >1 million read pairs per cell. The Ginkgo web-based algorithm, which relies on comparing the number of reads aligned to each genomic region, or “bin”, was used for quality control and CNV detection at varying bin sizes. We aimed to detect somatic CNVs, and determine if a known germline CNV (405 kb duplication) was visible at the single-cell level.

Results: We obtained WGA products after LCM with two kits (RepliG, Picoplex), which we used further. We sequenced 42 cells (18 RepliG, 24 Picoplex). The “noise” caused by uneven amplification was lower with Picoplex. The known CNV was called in one cell, although data suggested its presence in others. Several possible somatic CNVs were detected. Some cells isolated by LCM had highly aberrant profiles, possibly sectioning artefacts.

Conclusion: We report WGA and shallow WGS for CNV detection from human nigral dopaminergic neurons. LCM and nuclear capture were both successful, although caveats of sectioning, and appropriate nuclear markers, need to be considered respectively. Picoplex is preferable for CNV detection, consistent with published reports. Detection of sub-megabase CNVs remains challenging, as does orthogonal validation of single-cell CNVs unique.

References: [1] Leija-Salazar M, Piette C, Proukakis C. Somatic mutations in neurodegeneration. Neuropathol Appl Neurobiol. 2018 Apr;44(3):267-285. [2] Mokretar K, Pease D, Taanman J-W, Soenmez A, Ejaz A, Lashley T, Ling H, Gentleman S, Houlden H, Holton JL, Schapira AH, Nacheva E, Proukakis C. Somatic copy number gains of α-synuclein (SNCA) in Parkinson’s disease and multiple system atrophy brains. Brain 2018; 141(8): 2419–2431.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/sequencing-of-single-human-nigral-dopaminergic-neurons-for-somatic-copy-number-variants/