Eftychia Dimitriadou

Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains

Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell’s copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.

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

Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cells alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones.

Copy Number Variation Analysis by Array Analysis of Single Cells Following Whole Genome Amplification

Whole genome amplification is required to ensure the availability of sufficient material for copy number variation analysis of a genome deriving from an individual cell. Here, we describe the protocols we use for copy number variation analysis of non-fixed single cells by array-based approaches following single-cell isolation and whole genome amplification. We are focusing on two alternative protocols, an isothermal and a PCR-based whole genome amplification method, followed by either comparative genome hybridization (aCGH) or SNP array analysis, respectively.

Reciprocal 22q11.2 Deletion and Duplication in Siblings with Karyotypically Normal Parents.

The 22q11.2 locus is known to harbor a high risk for structural variation caused by non-allelic homologous recombination, resulting in deletions and duplications. Here, we describe the first family with one sibling carrying the 22q11 deletion and the other carrying the reciprocal duplication. FISH and SNP array analysis of the parents show a maternal origin for both deletion and duplication, without indications of balanced deletions/duplications or mosaicism. We hypothesize that germline mosaicism in the mother underlies the deletion and duplication, which would implicate a high recurrence risk for her offspring.