Single-Cell, Genome-wide Sequencing Identifies Clonal Somatic Copy-Number Variation in the Human Brain

Abstract

SUMMARYDe novo copy-number variants (CNVs) can cause neuropsychiatric disease, but the degree to which they occur somatically, and during development, is unknown. Single-cell whole-genome sequencing (WGS) in >200 single cells, including >160 neurons from three normal and two pathological human brains, sensitively identified germline trisomy of chromosome 18 but found most (≥95%) neurons in normal brain tissue to be euploid. Analysis of a patient with hemimegalencephaly (HMG) due to a somatic CNV of chromosome 1q found unexpected tetrasomy 1q in ~20% of neurons, suggesting that CNVs in a minority of cells can cause widespread brain dysfunction. Single-cell analysis identified large (>1 Mb) clonal CNVs in lymphoblasts and in single neurons from normal human brain tissue, suggesting that some CNVs occur during neurogenesis. Many neurons contained one or more large candidate private CNVs, including one at chromosome 15q13.2-13.3, a site of duplication in neuropsychiatric conditions. Large private and clonal somatic CNVs occur in normal and diseased human brains.

A generic, cost-effective, and scalable cell lineage analysis platform

“…Most of the MS panel is designed for (A) MSs of type AC on the X Chromosome to allow for monoallelic MS calling Wasserstrom et al 2008;Reizel et al 2011;Segev et al 2011;Shlush et al 2012) and for (B) the longest MS loci possible, which exhibit a higher mutation rate in vivo (Ellegren 2004;Supplemental Table S2). Notably, primers were designed such that the entire MS will be covered within a 150-bp Examples of single-cell analyses to reconstruct clonal/lineage analysis Wang et al 2012;Xu et al 2012;Gawad et al 2014;Lohr et al 2014;Lodato et al 2015) (Navin et al 2011;Cai et al 2014;Wang et al 2014) ) Salipante et al 2010;Reizel et al 2011Reizel et al , 2012Shlush et al 2012;Evrony et al 2015) read (see Methods). (2) We modified the second PCR to apply a sample barcode by utilizing combinations of forward and reverse PCR primer combinations, resulting in a dual indexed NGS library (Supplemental Fig.…”

CIN and Aneuploidy: Different Concepts, Different Consequences

“…These studies suggest that the level of aneuploidy in brain and liver is much lower than previously reported, with aneuploidy only affecting $2-5% of the cells, similar to the low rates of aneuploidy which were reported for epidermis. [62][63][64] Together these data suggest that many human tissues, including brain, skin and liver, contain a low level of aneuploidy. However, the exact contribution of aneuploid cells to in vivo tissues still needs to be more accurately quantified.…”

Construction of thousands of single cell genome sequencing libraries using combinatorial indexing

“…Estimates of the frequency of aneuploidy and large-scale copy number variation in the mammalian brain have varied widely, spanning a range of <5% to 33% [1][2][3][4] . This uncertainty largely stems from the inability to profile sufficient numbers of single cells to produce quantitative measurements.…”

“…The booming field of single cell sequencing continues to shine light on the abundance and breadth of genomic heterogeneity between cells in a variety of contexts, including somatic gains or losses of megabasepair-sized regions of the genome in the mammalian brain [1][2][3][4] , and tumor heterogeneity and clonal evolution [5][6][7] . Single cell genome sequencing studies have taken one of two approaches: high depth of sequencing per cell for purposes of single nucleotide variant detection 2,8 , or low-pass sequencing to identify copy number variants (CNVs) and the presence of aneuploidy 1,9,10 .…”

“…Single cell genome sequencing studies have taken one of two approaches: high depth of sequencing per cell for purposes of single nucleotide variant detection 2,8 , or low-pass sequencing to identify copy number variants (CNVs) and the presence of aneuploidy 1,9,10 . In the latter approach, the lack of a method to cost-effectively produce high numbers of single cell libraries has prevented the ability to quantitatively measure the frequency of CNV-harboring cells at population-level scale, or provide a robust analysis of heterogeneity in the context of cancer 11 .…”