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.

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    • "Next-generation whole-genome DNA sequencing (NGS or DNA-Seq) has also provided an additional platform besides qPCR and array-based methods to assess aneuploidy comprehensively in human embryos, including at the single-cell level (Hou et al. 2013; Fiorentino et al. 2014; Wang et al. 2014). Since aCGH covers approximately 30 % of the genome and can only detect gross chromosomal abnormalities at a lower threshold of approximately 1 MB (Bi et al. 2012), sub-chromosomal aberrations that are smaller than this resolution and missed by aCGH would be detectable by NGS in theory. In addition to examining copy number variation at a single nucleotide level, DNA-seq is also more cost-effective than aCGH since 48-96 samples or more can be simultaneously sequenced with high coverage by multiplexing with barcoded adapters. "
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    • "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]. "
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    • "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. "
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