Assessment of megabase-scale somatic copy number variation using single-cell sequencing

Abstract

Megabase-scale copy number variants (CNVs) can have profound phenotypic consequences. Germline CNVs of this magnitude are associated with disease and experience negative selection. However, it is unknown whether organismal function requires that every cell maintain a balanced genome. It is possible that large somatic CNVs are tolerated or even positively selected. Single-cell sequencing is a useful tool for assessing somatic genomic heterogeneity, but its performance in CNV detection has not been rigorously tested. Here, we develop an approach that allows for reliable detection of megabase-scale CNVs in single somatic cells. We discover large CNVs in 8%-9% of cells across tissues and identify two recurrent CNVs. We conclude that large CNVs can be tolerated in subpopulations of cells, and particular CNVs are relatively prevalent within and across individuals.[Supplemental material is available for this article.]Copy number variants (CNVs) can range in size from hundreds to millions of base pairs. Copy number changes affect approximately seven times as many base pairs as single-nucleotide variants and are major contributors to inter-individual differences (Sudmant et al. 2015). More than 65% of individuals harbor a germline CNV of at least 100 kb, and at least 1% of individuals have a CNV exceeding 1 Mb (Itsara et al. 2009). Although megabase-scale CNVs could be considered collectively common, the specific CNVs themselves are rare and often associated with disease (Girirajan et al. 2011). Not surprisingly, large CNVs experience negative selection, and their existence in a population is largely due to de novo events (Itsara et al. 2010).Although germline, megabase-scale CNVs are found in 1% of individuals, the prevalence of somatic CNVs is only beginning to be investigated. Array-based analyses of populations of cells from many individuals provided initial insight into this question. These studies identified megabase-scale somatic aberrations in up to 4% of individuals; however, the sensitivity was limited to CNVs present in >5% of cells (Forsberg et al. 2012;Jacobs et al. 2012;Laurie et al. 2012). These studies are thus blind to alterations that arise late in development or adversely affect fitness, as this would limit their propagation in a cell population. With the emergence of methods to amplify the genome of a single cell, single-cell sequencing now provides an alternate means of assessing the prevalence of somatic CNVs and offers the advantage of detecting variants that exist in as few as one cell. Recently, two groups performed low-coverage sequencing of single human neurons and reported at least one megabase-scale CNV in >40% of neurons (McConnell et al. 2013;Cai et al. 2014). These findings suggest much greater tolerance of large somatic CNVs compared to germline CNVs and raise the interesting possibility that somatic genomic heterogeneity contributes to phenotypic diversity within a tissue. However, it is still unclear how CNV detection methods perform when applied to individual cells, as single-cell sequ...

Sequencing thousands of single-cell genomes with combinatorial indexing

“…Studies have taken one of two approaches: high depth of sequencing per cell for single nucleotide variant detection 2,9 , or low-pass sequencing to identify copy number variants (CNVs) and aneuploidy 1,10,11 . In the latter approach, the lack of an efficient, cost-effective method to produce large numbers of single cell libraries has made it difficult to quantify the frequency of CNV-harboring cells at population scale, or to provide a robust analysis of heterogeneity in the context of cancer 12 .…”

Intersection of diverse neuronal genomes and neuropsychiatric disease: The Brain Somatic Mosaicism Network

“…The first single-cell study of neuronal CNVs analyzed 110 human frontal cortex neurons and found that 13 to 41% of the neurons contained at least one megabase-scale de novo CNV (6). Additional studies, which analyzed fewer neuronal genomes, confirmed that de novo CNVs occur in at least 10% of neurons (7, 8). CNVs can be shared by multiple neurons and inherited in a clonal manner (8).…”

“…Similarly, inversions can be identified through differences in the orientations of paired-end sequencing reads. Numerous approaches have been developed to identify CNVs from WGS (7, 87–89), and most can be applied directly to identify somatic mutations. For example, recent studies using WGA in conjunction with WGS have identified megabase-scale de novo CNVs in human and mouse neurons based on differences in read-depth across genomic bins (6–9).…”

Building a lineage from single cells: genetic techniques for cell lineage tracking

“…Large subchromosomal somatic CNVs can be detected in normal skin 82,83 and brain 83–85 , and these studies report large proportions of skin cells and neurons, approximately 30–70%, that contain at least one somatic CNV, including a small number of shared CNVs that arose during development 85 . Furthermore, the analysis of clonal CNVs can also illuminate genes and lineages that are responsible for disease; for example, brain tissue from patients with hemimegalencephaly contains neurons with somatic copy-number gains of chromosome 1q (containing the growth-promoting gene AKT3 ) 85 .…”