Application Note
Mosaicism between Single Neurons

Analysis of Somatic SNV and Indels in Single Neurons using
ResolveDNA Whole Genome Amplification.
Commentary by:

Jon Zawistowski, PhD

Senior Director
Research & Development
BioSkryb Genomics

Gary Harton, PhD

Chief Science Officer
BioSkryb Genomics

Accumulation of neuronal mutations over the lifetime of individual cells is linked to thedevelopment of neurological pathologies including epilepsy, dementia, Alzheimer's, as wellas anxiety and depression1. Accurately characterizing genomic mosaicism of single neurons
may ultimately revolutionize our understanding of brain development and function2,3. In thismanuscript, the authors used ResolveDNA Whole Genome Amplficiation (WGA) which utlizes
Primary Template-directed Amplification4 (PTA) to amplify single neurons and take advantage ofthe 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.

Introduction
Human life, beginning with a single cell, from asingle genome, undergoes nearly a trillion cell
divisions. As cell differentiation progresses fromthe embryo a wide variety of cell types emerge,
each phenotype with a specific function or job.
Through these divisions and differentiation,
errors occur in the replication and in the divisionof the daughter genomes. In contrast to previous
assumption that the brain operates from a
unitary genome, recent evidence confirms2,3,6
the hypothesis that somatic genetic diversity isprevalent in the human brain7.
While the ability to detect this variation has
advanced, a major bottleneck has been thedifficulty of amplifying the genome of a single

cell accurately and evenly so that it can bedelineated by Next Generation Sequencing(NGS). In a manuscript published by Luquette,
et al., previous methods for WGA, such as
multiple displacement amplification (MDA),were compared to ResolveDNA PTA at the single
neuron level.
In the manuscript, MDA was found to produce
non-uniformity across the genome andoften amplify homologous chromosomes of
diploid cells at different rates, leading to allelicimbalance. To capture these genetic changes
with greater precision, improved methods toaccurately amplify the genome were tested
By combining the high allelic balance, anduniform amplification of the ResolveDNA
system for single cell sequencing with the next
For Research Use Only. Not for use in diagnostic procedures.

Ultraspecific Somatic Mutation Detection in Single Neurons

Figure 1. Allelic Balance of PTA compared. Allele balance for germline
heterozygous SNPs in each sample. Each line corresponds to one single cell.
Values near 0.5 indicate balanced amplification of homologous chromosomes;
values near 0 or 1 indicate complete dropout of one allele.

generation computational analysis tool, Single Cell Analysis
or SCAN2, Luquette, et al. demonstrate the ability to provide
ultraspecific somatic SNV and indel detection in single neurons5.
The study performed whole-genome and SCAN2 analysis of
76 single prefrontal cortex neurons from eight neurotypicalindividuals while developing SCAN2, to more accurately
identify both clonal and non-clonal somatic (i.e., limited to asingle neuron) SNVs and indels using single cell sequencing
data from ResolveDNA amplified genomes.
The data generated from single neurons published in themanuscript reveal the allelic balance following WGA with
ResolveDNA is superior compared to previous work conductedin their laboratory using MDA (Figure 1). Allelic balancemeasures the evenness of amplification between alleles in adiploid cell; values near 0.5 indicate successful amplification ofboth alleles while values near 0 or 1 indicate loss of one allele.
The data in the manuscript show that single cell genomes
generated by ResolveDNA have allelic balance approaching
50% for germline heterozygous single nucleotide variants,
compared to MDA which has been noted to be more variable
in terms of allelic balance within and between individual cells
analyzed at the same time.
To further define the uniformity of genome coverage, Luquette
et al. used the MAPD metric to calculate regional amplification
bias (Figure 2). On average, only 37% of MDA-amplified
genomes exhibited balance levels in the range of 0.3-0.7
compared to 68% of PTA genomes. The authors also found therate of amplification failure among ResolveDNA reactions tobe low. Only a single neuron showed evidence of amplification
failure in the form of near-complete loss of several haplotypes.
The data indicate that ResolveDNA has roughly a 3X reductionin MAPD, which represents a dramatic improvement in coverageand amplification uniformity.

Figure 2. MAPD of PTA compared. Median absolute pairwise deviation(MAPD) for MDA amplified and PTA-amplified neuronal genomes from thesame individuals; lower values indicate better performance. The average
MAPDs of MDA (0.75) and PTA (0.21) correspond to an average fluctuationin read depth between neighboring 50 kb windows of 68% and 14%,respectively.

A demonstrative example of the improved uniformity obtainedwith ResolveDNA PTA relative to MDA is the measurement of CNV
in single neuron genomes (Figure 3). The data demonstrates thatuniformity and balance across the genome is superior to thatfound employing MDA. These improvements in whole genome
amplification uniformity led to dramatic improvements in thecharacterization of genetic mosaicism in the brain. Specifically,reported in the Luquette et al. manuscript, 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. In addition, somatic indels also increase
by at least 2 indels per year per neuron which may have a moresignificant impact on gene function than somatic SNVs inhuman neurons.

Figure 3. Allelic Balance of PTA compared. Single neurons were collected fromthe prefrontal cortices of brains of 8 individuals ranging in age from infantile
to elderly. Single neurons were amplified by either PTA or MDA and thensequenced. Representative copy number profiles for bulk (top), MDA-amplified(middle) and PTA-amplified (bottom) genomes.
For Research Use Only. Not for use in diagnostic procedures.

Ultraspecific Somatic Mutation Detection in Single Neurons

Summary
In the manuscript by Luquette et al., the authors combine theResolveDNA single cell genome amplification system with thenovel development of SCAN2 to demonstrate the ability todelineate single nucleotide variation with the highest levelsof precision from single neurons. The data demonstrates thatfurther refinement of the combined methods may lead tonew biological hypotheses that bring about a redefinition of
the underlying genomic mechanisms that drive neurologicaldysfunction and pathology. The Luquette, etal. manuscript
clearly defines that both the methods employed to perform
template amplification and the process to interpret theresultant next generation sequencing data are critical to ensure
the highest degree of confidence in conclusions drawn fromsuch experiments. Continuous development of methods thatrefine the analysis process are essential to the expanding single
cell genomic field and are imperative across various fieldsof study. Including, but not limited to, neurology, oncology,reproductive medicine and studies of the microbiome.

References:
1.

Proukakis, C., Somatic mutations in neurodegeneration: An
update. Neurobiol Dis, 2020. 144: p. 105021.

2.

Evrony, G.D., One brain, many genomes. Science, 2016. 354(6312):
p. 557-558.

3.

Evrony, G.D., et al., Resolving rates of mutation in the brain using
single-neuron genomics. Elife, 2016. 5.

4.

Gonzalez, V., et al., Accurate Genomic Variant Detection in Single
Cells with Primary Template-Directed Amplification. 2020: www.
biorxiv.org. p. 1-53.

5.

Luquette, L., et al., Ultraspecific somatic SNV and indel detection
in single neurons using primary template-directed amplification.
bioRxiv, 2021.

6.

Lodato, M.A., et al., Aging and neurodegeneration are associatedwith increased mutations in single human neurons. Science, 2018.
359(6375): p. 555-559.

7.

Muotri, A.R. and F.H. Gage, Generation of neuronal variability andcomplexity. Nature, 2006. 441(7097): p. 1087-93.

8.

BioSkryb Genomics, i., ResolveDNA™ Whole Genome
Amplification Kit For high-quality single-cell and low-input DNA
amplification, in www.bioskryb.com, B. Genomics, Editor. 2021,
BioSkryb Genomics: Durham, NC. USA

Note:This application note reviews and details a manuscript published as apreprint on BioRxiv as follows:
LJ Luquette, MB Miller, Z Zhou, CL Bohrson, A Galor, MA Lodato, CGawad, J West, CA Walsh, PJ Park. Ultraspecific somatic SNV andindel detection in single neurons using primary template-directed
amplification. doi:https://doi.org/10.1101/2021.04.30.44203 (https://www.biorxiv.org/content/10.1101/2021.04.30.442032v1)

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TAS-023, 01/2022
For Research Use Only. Not for use in diagnostic procedures.