Single-cell paired-end genome sequencing reveals structural variation per cell cycle

Abstract

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis.

Low-grade chromosomal mosaicism in human somatic and embryonic stem cell populations

“…It is important to bear in mind that these studies are based on the analysis of DNA from extractions from large numbers of cells, which only allows detection of CNVs if they are present in at least 5-10% of the cells 11,12 . In contrast, the recent development of new methods for the comprehensive study of the single cell genome [13][14][15][16] , epigenome 17 and transcriptome 18 is leading to a new understanding of cellular diversity. For instance, the study of single breast cancer cells revealed a significant number of de novo DNA copy number changes that appeared after only one cell cycle 15 and the analysis of large numbers of single cells has provided interesting insight in the evolution of genetically different cell populations within one tumour [14][15][16] .…”

“…In contrast, the recent development of new methods for the comprehensive study of the single cell genome [13][14][15][16] , epigenome 17 and transcriptome 18 is leading to a new understanding of cellular diversity. For instance, the study of single breast cancer cells revealed a significant number of de novo DNA copy number changes that appeared after only one cell cycle 15 and the analysis of large numbers of single cells has provided interesting insight in the evolution of genetically different cell populations within one tumour [14][15][16] . In the field of hiPSC, a recent study described low-grade mosaicism in neurons and differentiated hiPSCs at high resolution using array-based comparative genomic hybridization (aCGH) and single-cell sequencing 19 .…”

Concurrent Whole-Genome Haplotyping and Copy-Number Profiling of Single Cells

“…To determine the accuracy of copy-number profiling, we computed the distances between (1) the a priori known t(1;16) translocation breakpoint on chromosome 16 of family PGD004, which was determined to base resolution using single-cell paired-end sequencing and further validated by Sanger sequencing, 20 and (2) the copy-number breakpoints-that result from the unbalanced inheritance of the derivative chromosomes of t(1;16)-detected in the single blastomeres after siCHILD analysis of the SNP logR values.…”

“…This is because the underlying copy-number state of the SNP is ignored and because the abundant WGA artifacts in single-cell assays produce false homozygous and heterozygous SNP calls. 13,14 Various methods for DNA copy-number profiling of single cells have been developed and rely on transforming probe intensities of microarrays 3,10,[15][16][17] or next-generation sequence read counts [18][19][20][21] into DNA copy numbers.…”

“…22,23 Whereas deletions can be confirmed by loss of heterozygosity across SNPs over a longer distance, 15 discrete SNP-genotype calls nor regular SNP B-allele fractions can effectively validate duplications in single cells. 20 Additionally, resolving the mitotic and meiotic origin as well as the parental origin of DNA anomalies in single cells, or determining the ploidy of the cell, is not straightforward. 17,24 Although in theory the analysis of SNP B-allele fractions (BAFs)-i.e., the frequency with which a SNP variant allele occurs in the dataset of a DNA sample-should enable the determination of haplotypes and their underlying copynumber state, this has remained impossible at the singlecell level because single-cell analyses require WGA, a process known to introduce (stochastic) allelic distortions due to amplification artifacts.…”