Detection of=1?Mb microdeletions and microduplications in a single cell using custom oligonucleotide arrays

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
Prenatal Diagnosis (Impact Factor: 3.27). 01/2012; 32(1):10-20. DOI: 10.1002/pd.2855
Source: PubMed


High resolution detection of genomic copy number abnormalities in a single cell is relevant to preimplantation genetic diagnosis and potentially to noninvasive prenatal diagnosis. Our objective is to develop a reliable array comparative genomic hybridization (CGH) platform to detect genomic imbalances as small as ~1Mb ina single cell.
We empirically optimized the conditions for oligonucleotide-based array CGH using single cells from multiple lymphoblastoid cell lines with known copy number abnormalities. To improve resolution, we designed custom arrays with high density probes covering clinically relevant genomic regions.
The detection of megabase-sized copy number variations (CNVs) in a single cell was influenced by the number of probes clustered in the interrogated region. Using our custom array, we reproducibly detected multiple chromosome abnormalities including trisomy 21, a 1.2Mb Williams syndrome deletion, and a 1.3Mb CMT1A duplication. Replicate analyses yielded consistent results.
Aneuploidy and genomic imbalances with CNVs as small as 1.2Mb in a single cell are detectable by array CGH using arrays with high-density coverage in the targeted regions. This approach has the potential to be applied for preimplantation genetic diagnosis to detect aneuploidy and common microdeletion/duplication syndromes and for noninvasive prenatal diagnosis if single fetal cells can be isolated.

Download full-text


Available from: Sau Wai Cheung, Oct 10, 2015
1 Follower
118 Reads
  • Source
    • "Standard DNA-microarrays can detect copy number variations (CNVs) larger than 2.5 Mb from a single-cell genome [20]–[22], while targeted array comparative genomic hybridizations can discover approximately 1 Mb-sized DNA imbalances [23], although remarkably, CNVs as small as 56 kb in single-cell PCR-based WGA products have been detected [24]. Similarly, SNP-arrays can find copy number aberrations encompassing millions of bases in a cell [25]–[28], but have the advantage of enabling the discovery of copy neutral DNA anomalies and regions of loss-of-heterozygosity (LOH), and allow inferring genome-wide haplotypes [29]–[31]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Advances in whole-genome and whole-transcriptome amplification have permitted the sequencing of the minute amounts of DNA and RNA present in a single cell, offering a window into the extent and nature of genomic and transcriptomic heterogeneity which occurs in both normal development and disease. Single-cell approaches stand poised to revolutionise our capacity to understand the scale of genomic, epigenomic, and transcriptomic diversity that occurs during the lifetime of an individual organism. Here, we review the major technological and biological breakthroughs achieved, describe the remaining challenges to overcome, and provide a glimpse into the promise of recent and future developments.
    PLoS Genetics 01/2014; 10(1):e1004126. DOI:10.1371/journal.pgen.1004126 · 7.53 Impact Factor
  • Source
    • "The third major improvement could be achieved by usage of high-resolution aCGH arrays based on oligonucleotides. Studies published in the recent years indicated applicability of the new generation of high-resolution SNP and CGH arrays for single-cell CGH analysis [26], [35], [39]–[43]. In comparison to the previously used BAC-based arrays [24], [25], [44] these technologies offer lower qualitative variability of the array slide manufacturing process, customizable microarray designs and very low probe spacing. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Disseminated cancer cells (DCCs) and circulating tumor cells (CTCs) are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape. Using the Ampli1™ whole genome amplification (WGA) technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points. The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.
    PLoS ONE 01/2014; 9(1):e85907. DOI:10.1371/journal.pone.0085907 · 3.23 Impact Factor
  • Source
    • "Navin and colleagues [54,55] showed that low-coverage single-end sequencing after PCR-based WGA enabled profiling a cell's DNA copy-number landscape at a resolution unattainable with even the highest-resolution array approaches available [37,56]. They developed a methodology using focal read depth analysis, in which the number of single-end reads mapping uniquely to specific bins across the human reference genome is counted and transformed to a copy-number state per bin. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Preimplantation genetic diagnosis (PGD) aims to help couples with heritable genetic disorders to avoid the birth of diseased offspring or the recurrence of loss of conception. Following in vitro fertilization, one or a few cells are biopsied from each human preimplantation embryo for genetic testing, allowing diagnosis and selection of healthy embryos for uterine transfer. Although classical methods, including single-cell PCR and fluorescent in situ hybridization, enable PGD for many genetic disorders, they have limitations. They often require family-specific designs and can be labor intensive, resulting in long waiting lists. Furthermore, certain types of genetic anomalies are not easy to diagnose using these classical approaches, and healthy offspring carrying the parental mutant allele(s) can result. Recently, state-of-the-art methods for single-cell genomics have flourished, which may overcome the limitations associated with classical PGD, and these underpin the development of generic assays for PGD that enable selection of embryos not only for the familial genetic disorder in question, but also for various other genetic aberrations and traits at once. Here, we discuss the latest single-cell genomics methodologies based on DNA microarrays, single-nucleotide polymorphism arrays or next-generation sequence analysis. We focus on their strengths, their validation status, their weaknesses and the challenges for implementing them in PGD.
    Genome Medicine 08/2013; 5(8):71. DOI:10.1186/gm475 · 5.34 Impact Factor
Show more