Christopher Walsh

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PTA is our method of choice if you want reproducible, efficient, and evenly amplified genomes from single cells for single nucleotide variant detection."

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Bullard Professor of Neurology, Harvard Medical School Chief Division of Genetics, Boston Children’s Hospital Investigator, Howard Hughes Medical Institute

## Why invest in ultra-deep bulk sequencing if you can accurately detect the rarest of somatic mutations in single neurons?

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The nervous system is comprised of morphologically and functionally diverse neurons to support a web of unique neural networks. It is now known that individual neurons are also genetically heterogeneous, accumulating somatic mutations during development and aging. This mosaicism impacts cognition and behavior, and contributes to neurological disorders such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, epilepsy, and stroke.

Unlike cancer mutations, somatic mutations in neural tissues are often non-clonal. Ultra-deep bulk sequencing of very small tissue samples offers one approach for detecting and characterizing these unique mutations, but is technically challenging and can be cost-prohibitive.

BioSkryb’s highly uniform single-cell whole genome amplification ([WGA](/products/)) technology ([PTA](/technology/)) eliminates the allelic imbalance and artifacts resulting from other WGA methods such as multiple displacement amplification (MDA). Combined with next-generation computational tools (such as SCAN2, see manuscript below), PTA enables the accurate and precise detection of both clonal and non-clonal somatic single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number variation (CNV) in the genomes of single neurons. This represents a technological breakthrough in elucidating the enigma of brain and other neural mosaicism, ultimately supporting improved diagnosis and personalized treatment.

Primary template-directed amplification (PTA) is an improved amplification technique for single-cell DNA sequencing. We generated whole-genome analysis of 76 single neurons and developed SCAN2, a computational method to accurately identify both clonal and non-clonal somatic (i.e., limited to a single neuron) single nucleotide variants (SNVs) and small insertions and deletions (indels) using PTA data. Our analysis confirms an increase in non-clonal somatic mutation in single neurons with age, but revises estimates for the rate of this accumulation to be 15 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels also increase by at least 2 indels per year per neuron and that indels may have a larger impact on gene function than somatic SNVs in human neurons.

Here we compare single neurons amplified by both the MDA and PTA protocols from the prefrontal cortices of the same individuals and find that PTA substantially improves upon MDA

Ultraspecific somatic SNV and indel detection in single neurons using primary template-directed amplification

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Accumulation of neuronal mutations over the lifetime of individual cells is linked to the development of neurological pathologies including epilepsy, dementia, Alzheimer's, as well as anxiety and depression1. Accurately characterizing genomic mosaicism of single neurons may ultimately revolutionize our understanding of brain development and function2,3. In this manuscript, the authors used ResolveDNA Whole Genome Amplficiation (WGA) which utlizes Primary Template-directed Amplification4 (PTA) to amplify single neurons and take advantage of the high allelic balance provided by ResolveDNA WGA, combined with their proprietary software analysis tool named SCAN2. This bioinformatics algorithm is a novel single-cell computational analysis tool that was paired with PTA to delineate single-cell mosaicism within the brain. ResolveDNA provides broad amplification across the entire genome with excellent uniformity, which allows for unprecedented sensitivity and specificity for detecting single nucleotide variation (SNV), insertions and deletion (Indels), structural variations, and copy number variations (CNV)5.

Authors combine the ResolveDNA single cell genome amplification system with the novel development of SCAN2 to demonstrate the ability to delineate single nucleotide variation with the highest levels of precision from single neurons.

Analysis of Somatic SNV and Indels in Single Neurons using ResolveDNA Whole Genome Amplification

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Dementia in Alzheimer’s disease progresses alongside neurodegeneration1–4, but the specific events that cause neuronal dysfunction and death remain poorly understood. During normal ageing, neurons progressively accumulate somatic mutations5 at rates similar to those of dividing cells6,7 which suggests that genetic factors, environmental exposures or disease states might influence this accumulation5. Here we analysed single-cell whole-genome sequencing data from 319 neurons from the prefrontal cortex and hippocampus of individuals with Alzheimer’s disease and neurotypical control individuals. We found that somatic DNA alterations increase in individuals with Alzheimer’s disease, with distinct molecular patterns. Normal neurons accumulate mutations primarily in an age-related pattern (signature A), which closely resembles ‘clock-like’ mutational signatures that have been previously described in healthy and cancerous cells6–10. In neurons affected by Alzheimer’s disease, additional DNA alterations are driven by distinct processes (signature C) that highlight C>A and other specific nucleotide changes. These changes potentially implicate nucleotide oxidation4,11, which we show is increased in Alzheimer’s-disease-affected neurons in situ. Expressed genes exhibit signature-specific damage, and mutations show a transcriptional strand bias, which suggests that transcription-coupled nucleotide excision repair has a role in the generation of mutations. The alterations in Alzheimer’s disease affect coding exons and are predicted to create dysfunctional genetic knockout cells and proteostatic stress. Our results suggest that known pathogenic mechanisms in Alzheimer’s disease may lead to genomic damage to neurons that can progressively impair function. The aberrant accumulation of DNA alterations in neurodegeneration provides insight into the cascade of molecular and cellular events that occurs in the development of Alzheimer’s disease.

Here we analysed single-cell whole-genome sequencing data from 319 neurons from the prefrontal cortex and hippocampus of individuals with Alzheimer’s disease and neurotypical control individuals.

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Somatic genomic changes in single Alzheimer’s disease neurons

Primary Template-directed Amplification or PTA employs controlled reaction parameters to reproducibly recover greater than 95% of the genome of single-cells and severely limited input samples with best-in-class fidelity and uniformity. 

Discover the difference BioSkryb Genomics can make for you in WGA

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Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

Accurate Genomic Variant Detection in Single Cells with Primary Template-Directed Amplification

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Somatic mutations have causative roles in many diseases and contribute to neuropsychiatric disorders. Here, we analyzed 131 human brains (44 neurotypical, 19 with Tourette syndrome, 9 with schizophrenia, and 59 with autism) for somatic single nucleotide and structural mutations after whole genome sequencing to a depth of over 200X. Typically, brains had 20 to 60 detectable single nucleotide mutations that likely arose in early development; however, about 5% of brains harbored hundreds and up to 2000 somatic mutations. Hypermutability was associated with age and putative damaging mutations in genes previously implicated in cancer and likely reflects in vivo clonal expansions. Somatic duplications of likely early developmental origin were present in 1 out of 20 normal and diseased brains, reflecting background mutagenesis. Brains of individuals with autism were enriched in somatic deletions and in point mutations that create putative transcription factor binding motifs in enhancers that are active in the developing brain. The most affected motifs corresponded to MEIS transcription factors, suggesting a potential link between their involvement in gene regulation and autism.

Our study revealed a complex etiology of somatic mutations in brain and their possible relationship with phenotype.

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Somatic mutations reveal hypermutable brains and are associated with neuropsychiatric disorders

Characterizing the mechanisms of somatic mutations in the brain is important for understanding aging and disease, but little is known about the mutational patterns of different cell types. We performed whole-genome sequencing of 71 oligodendrocytes and 51 neurons from neurotypical individuals (0.4 to 104 years old) and identified >67,000 somatic single nucleotide variants (sSNVs) and small insertions and deletions (indels). While both cell types accumulate mutations with age, oligodendrocytes accumulate sSNVs 69% faster than neurons (27/year versus 16/year) whereas indels accumulate 42% slower (1.8/year versus 3.1/year). Correlation with single-cell RNA and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These patterns highlight differences in the mutagenic processes in glia and neurons and suggest cell type-specific, age-related contributions to neurodegeneration and oncogenesis.

In this study, we assessed genome-wide rates and patterns of aging-related somatic mutations in OLs compared to neurons isolated from the same individuals with single-cell whole-genome sequencing (scWGS).

Neurology

Why invest in ultra-deep bulk sequencing if you can accurately detect the rarest of somatic mutations in single neurons?

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Christopher Walsh