Somatic CNV Detection by Single-Cell Whole-Genome Sequencing in Postmortem Human Brain

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• Growing evidence links somatic mutations, including copy-number variants (CNVs), to neurodegenerative diseases.
• Understanding the precise role, origin, and impact of these mutations on disease progression remains a significant challenge.
• Single-cell whole-genome sequencing (scWGS) offers a powerful approach to investigate cellular heterogeneity in mutation profiles.

Purpose of the Study:

• To provide detailed laboratory and bioinformatic protocols for detecting megabase-scale CNVs in single human brain cells.
• To apply these methods to postmortem brain samples from patients with multiple system atrophy (MSA).
• To compare different whole-genome amplification (WGA) chemistries for scWGS applications.

Main Methods:

• Development and application of protocols for immunolabeling and nuclei selection for whole-genome amplification (WGA).
• Utilizing single-cell whole-genome sequencing (scWGS) to detect megabase-scale copy-number variants (CNVs).
• Comparison of Multiple Displacement Amplification (MDA) and PicoPLEX WGA chemistries using control substantia nigra samples.

Main Results:

• Successful detection of megabase-scale CNVs in single cells from human MSA postmortem brains.
• Presentation of a comparative analysis of MDA and PicoPLEX WGA methods for scWGS.
• Demonstration of the protocol's applicability to specific cell types relevant to synucleinopathies, such as dopaminergic neurons and oligodendrocytes.

Conclusions:

• The presented protocols enable robust detection of large-scale CNVs in single brain cells.
• scWGS is a valuable tool for investigating the contribution of somatic mutations to neurodegeneration.
• The methodology can be adapted for various tissue and cell types to study genetic alterations in disease.