Genetic mosaicism, the presence of genetically distinct cell populations within an individual, arises due to somatic mutations occurring during embryogenesis and throughout life. This phenomenon is critical in both normal development and disease pathogenesis, particularly in the aging brain. Understanding mosaicism requires precise genomic analysis, which has been significantly advanced by single-cell whole-genome sequencing (WGS). However, accurate detection of somatic mutations at the single-cell level has been hindered by technical limitations in whole-genome amplification (WGA). Recent advances, including primary template-directed amplification (PTA), have greatly improved the fidelity of single-cell genomics.

Origins of Genetic Mosaicism

Mosaicism occurs when mutations arise in a subset of cells during early embryogenesis or later in life due to environmental stressors, replication errors, or DNA repair deficiencies. These mutations can be classified into several types:

  • Point mutations: Single nucleotide changes that may impact gene function.
  • Copy number variations (CNVs): Duplications or deletions of large genomic segments.
  • Structural variants: Large-scale genomic rearrangements such as translocations and inversions.
  • Mobile element insertions: Retrotransposon activity leading to new DNA insertions.

Early mutations in embryogenesis can lead to tissue-specific mosaicism, which has implications for developmental disorders and cancer predisposition. In the aging brain, accumulated somatic mutations contribute to neurodegenerative diseases and cognitive decline (Miller et al., 2021). Due to their low allele frequencies, detecting these mutations requires ultra-sensitive genomic techniques.

The Necessity of Single-Cell Whole-Genome Sequencing

Traditional bulk sequencing averages genetic information across many cells, obscuring rare somatic mutations. Single-cell WGS allows for precisely identifying these mutations in individual cells, which is crucial for studying genetic heterogeneity in tissues like the brain, where neurons exhibit high mutation burdens with age (Lodato et al., 2018).

To perform single-cell WGS, whole-genome amplification (WGA) is necessary to amplify the minute DNA content of a single cell (∼6 pg). However, conventional WGA methods introduce amplification bias and errors, limiting their effectiveness in detecting somatic mutations.

Technical Limitations of Whole-Genome Amplification

Early WGA methods suffered from uneven genome coverage and artifacts, complicating mutation detection. The three primary WGA techniques used for single-cell WGS are:

  1. PicoPlex (Takara Bio)
    • Uses preamplification followed by PCR amplification.
    • Strengths: Low-input DNA requirement and streamlined workflow.
    • Weaknesses: High allelic dropout and significant amplification bias (Takara Bio).
  2. REPLI-g (Qiagen)
    • Based on multiple displacement amplification (MDA) using phi29 polymerase.
    • Strengths: High coverage uniformity and minimal sequence bias.
    • Weaknesses: Prone to chimeric artifacts and poor amplification of GC-rich regions (Qiagen; Gonzalez-Pena et al., 2021).
  3. Primary Template-Directed Amplification (PTA) (BioSkryb)
    • Selectively amplifies original DNA templates rather than copied amplicons.
    • Strengths: Significantly reduces errors and amplification bias, leading to higher accuracy in single-cell mutation detection (BioSkryb Technologies, 2024).
    • Weaknesses: Slightly more expensive than REPLI-g.

Advances in Detecting Mosaicism with PTA

PTA has revolutionized single-cell WGS by maintaining high genome coverage while minimizing amplification errors. Compared to traditional WGA methods, PTA provides:

  • Higher sensitivity for detecting rare somatic mutations.
  • Improved uniformity across the genome, reducing false positives.
  • Better representation of GC-rich and repetitive regions.

 

 

Fig 1 (from Gonzalez-Pena PNAS 2021)

In a study by Gonzalez-Pena et al., the authors show that PTA significantly improved uniformity of coverage compared to MDA (Figure 1). They also demonstrate that PTA approaches the two bulk (unamplified) samples at every coverage depth, which is a significant improvement over traditional MDA, which maxes out at 75% of the genome.

These advancements have profound implications for studying neurodevelopmental disorders, cancer evolution, and aging-related somatic mutation accumulation (Bae et al., 2018).

Conclusion

Genetic mosaicism is critical in human development and aging, necessitating accurate detection methods. Single-cell WGS has emerged as a powerful tool, but its success depends on reliable whole-genome amplification. While PicoPlex and REPLI-g have contributed to early single-cell studies, PTA has set a new standard by significantly improving accuracy. As sequencing technologies evolve, these advancements will enhance our understanding of somatic mutations in health and disease. PTA kits can be purchased from BioSkryb Genomics or run using a PTA service provider, like MiRXES. MiRXES is one of the few service providers certified to run the ResolveOME Whole Genome and Transcriptome Amplification Kit. The ResolveOME Whole Genome and Transcriptome Amplification Kit combines whole-genome and full-transcript transcriptome sequencing from the same cell. MiRXES also offers spatial transcriptomic services using Stereo-seq to combine spatial information with gene expression data.

References

  1. Miller MB, Reed HC, Walsh CA. Brain Somatic Mutation in Aging and Alzheimer’s Disease. Annual Review of Genomics Human Genetics. 2021 Aug 31;22:239-256.
  2. Lodato, M.A., et al. (2018). “Aging and neurodegeneration are associated with increased mutations in single human neurons.” Science, 359(6375), 555-559.
  3. Gonzalez-Pena, V., et al. (2021). “Accurate genomic variant detection in single cells with primary template-directed amplification.” PNAS, Jun 15;118(24).
  4. Bae, T., et al. (2018). “Different mutational rates and mechanisms in human cells at different developmental stages.” Science, 359(6375):550-555..
  5. BioSkryb Technologies. (2024). “Primary template-directed amplification (PTA).” Retrieved from [https://www.bioskryb.com/technology/]

 

 

 

Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is the leading cause of dementia globally, affecting over 32 million people as of 2023. Characterized by cognitive decline, memory deficits, and behavioral changes, AD is marked by the accumulation of amyloid-β plaques and neurofibrillary tangles in the brain’s gray matter. Aging is a significant risk factor, particularly in the prefrontal cortex (PFC), a region critical for higher cognitive functions. Despite years of research, the molecular mechanisms distinguishing AD from normal aging remain elusive. In a recently published study titled “Stereo-seq of the prefrontal cortex in aging and Alzheimer’s disease,” researchers at Tulane University in the Deming Department of Medicine performed the first subcellular-resolution spatial transcriptome atlas of the PFC from Alzheimer’s disease patients. The researchers published their findings in the January 8th, 2025, issue of Nature Communications.

Background and Objectives

The researchers aimed to uncover the molecular mechanisms underlying aging-related susceptibility to AD by comparing transcriptomes from the PFC of six male AD patients and six age-matched controls. Previous studies, such as those using 10X Visium, revealed AD-associated gene expression changes but lacked the spatial resolution to dissect single-cell interactions. By employing Stereo-seq, this study sought to:

  1. Identify transcriptional differences across PFC layers.
  2. Characterize cell-cell interactions influencing AD pathology.
  3. Highlight potential therapeutic targets.

Methodology

Using Stereo-seq, the team analyzed cryosections of PFC tissue, focusing on seven predominant cell types: astrocytes (Ast), excitatory neurons (Ex), inhibitory neurons (Inh), microglia (Mic), endothelial cells (End), oligodendrocyte progenitor cells (Opc), and oligodendrocytes (Oli). By aligning transcriptional data with spatial maps, they generated a detailed atlas revealing layer-specific and cell-type-specific alterations in AD.

 

From Gong Y. et al. Nature Communications 2025

Key Findings

  1. Disruption of Laminar Structure The study identified significant structural changes in PFC layers, particularly in advanced AD cases. Layers II-VI showed marked thinning and transcriptional disruptions, highlighting the vulnerability of these regions to neurodegeneration.
  2. Gene Modules Linked to Neuroprotection
    • Immune Response and Inflammation Regulation: Genes regulating immune activity were upregulated in stressed neurons and glial cells, emphasizing the role of inflammation in AD progression.
    • Protein Homeostasis: Modules governing protein degradation—crucial for clearing amyloid-β and tau aggregates—were significantly downregulated.
    • Synaptic Function: Genes involved in synaptic transmission and plasticity showed decreased expression, correlating with cognitive decline in AD.
  3. ZNF460: A Potential Therapeutic Target The transcription factor ZNF460 was identified as a regulator of the aforementioned gene modules. Its downregulation in AD suggests it could be a promising therapeutic target for restoring neuroprotective pathways.
  4. Spatial Patterns of Stress Response Stressed neurons exhibited elevated mitochondrial gene expression, correlating with neuroinflammation and oxidative stress. Adjacent glial cells—particularly astrocytes—showed transcriptional changes supporting neuronal survival and amyloid-β clearance.
  5. Altered Cell-Cell Communication Communication networks between cortical layers, mediated by ligand-receptor interactions, deteriorated with AD progression. Notably, the loss of glutamate signaling in excitatory neurons underscored synaptic dysfunction as a hallmark of AD.

 

Implications for Alzheimer’s Research

This study provides critical insights into the spatial and molecular dynamics of AD in the PFC:

  • High-Resolution Mapping: By identifying transcriptional changes at single-cell resolution, researchers can pinpoint early markers of AD pathology.
  • Targeted Therapies: Discovering regulators like ZNF460 opens avenues for precision medicine aimed at restoring neuronal health.
  • Enhanced Understanding of Disease Progression: The spatial context of gene expression reveals how cellular interactions evolve in response to AD pathology, offering a more holistic view of the disease.

Expanding Access to Stereo-seq Technology

The accessibility of Stereo-seq services is crucial for advancing research. MiRXES, a leading genomics and bioinformatics service provider, was the first independent U.S.-based organization to offer Stereo-seq. In January 2025, MiRXES expanded its capabilities to include formalin-fixed, paraffin-embedded (FFPE) tissue, a common medium used for archived samples. This advancement significantly broadens the scope of AD research by enabling the study of historical tissue collections.

Conclusion

Integrating Stereo-seq into Alzheimer’s research marks a new era of discovery, offering unprecedented clarity into the disease’s cellular and spatial dynamics. By bridging the gap between molecular profiling and spatial context, this technology holds the potential to unravel the complexities of neurodegeneration and pave the way for innovative therapies. As accessibility to Stereo-seq services grows, the scientific community stands on the cusp of breakthroughs that could redefine our understanding and treatment of Alzheimer’s disease.

Keywords and abbreviations: Stereo-seq, spatial transcriptomics, Alzheimer’s Disease (AD), prefrontal Cortex (PFC), neurodegeneration, astrocytes (Ast), excitatory neurons (Ex), inhibitory neurons (Inh), microglia (Mic), endothelial cells (End), oligodendrocyte progenitor cells (Opc), oligodendrocytes (Oli), formalin-fixed paraffin-embedded (FFPE) tissue, Visium, dementia, genomics