Direct, genome-wide assessment of DNA mutations in single cells

Department of Genetics, Albert Einstein College of Medicine, New York, NY 10461, USA.
Nucleic Acids Research (Impact Factor: 9.11). 11/2011; 40(5):2032-40. DOI: 10.1093/nar/gkr949
Source: PubMed


DNA mutations are the inevitable consequences of errors that arise during replication and repair of DNA damage. Because of their random and infrequent occurrence, quantification and characterization of DNA mutations in the genome of somatic cells has been difficult. Random, low-abundance mutations are currently inaccessible by standard high-throughput sequencing approaches because they cannot be distinguished from sequencing errors. One way to circumvent this problem and simultaneously account for the mutational heterogeneity within tissues is whole genome sequencing of a representative number of single cells. Here, we show elevated mutation levels in single cells from Drosophila melanogaster S2 and mouse embryonic fibroblast populations after treatment with the powerful mutagen N-ethyl-N-nitrosourea. This method can be applied as a direct measure of exposure to mutagenic agents and for assessing genotypic heterogeneity within tissues or cell populations.

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    • "The proofreading capacity of φ29 improves sequence fidelity during WGA [18], [37], [38], [119]. Furthermore, MDA amplifies the majority of a cell's genome and appears a preferred method for SNP genotyping [15], [17], [18] or base mutation detection [18], [37], [38], [120], but ADO and PA occurs. Following MDA, single-cell copy number profiles can be distorted [15], [17] —although improvements are emerging [19] —and chimeric DNA amplification products are created [17], [121]. "
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    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.
    Full-text · Article · Jan 2014 · PLoS Genetics
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    • "Furthermore, the amount and nature of artifacts can vary significantly between different WGA methods. Although pure MDA-based WGA methods appear to be the method of choice for typing single-nucleotide variants (15–19), WGA-methods involving PCR seem to preserve single-cell DNA-copy number changes more accurately during the amplification process and may be used for single-nucleotide variant detection as well (20–22). "
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    ABSTRACT: Disseminated tumor cells (DTCs) detected in the bone marrow have been shown as an independent prognostic factor for women with breast cancer. However, the mechanisms behind the tumor cell dissemination are still unclear and more detailed knowledge is needed to fully understand why some cells remain dormant and others metastasize. Sequencing of single cells has opened for the possibility to dissect the genetic content of subclones of a primary tumor, as well as DTCs. Previous studies of genetic changes in DTCs have employed single-cell array comparative genomic hybridization which provides information about larger aberrations. To date, next-generation sequencing provides the possibility to discover new, smaller, and copy neutral genetic changes. In this study, we performed whole-genome amplification and subsequently next-generation sequencing to analyze DTCs from two breast cancer patients. We compared copy-number profiles of the DTCs and the corresponding primary tumor generated from sequencing and SNP-comparative genomic hybridization (CGH) data, respectively. While one tumor revealed mostly whole-arm gains and losses, the other had more complex alterations, as well as subclonal amplification and deletions. Whole-arm gains or losses in the primary tumor were in general also observed in the corresponding DTC. Both primary tumors showed amplification of chromosome 1q and deletion of parts of chromosome 16q, which was recaptured in the corresponding DTCs. Interestingly, clear differences were also observed, indicating that the DTC underwent further evolution at the copy-number level. This study provides a proof-of-principle for sequencing of DTCs and correlation with primary copy-number profiles. The analyses allow insight into tumor cell dissemination and show ongoing copy-number evolution in DTCs compared to the primary tumors.
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    • "Zong et al. [35] required data from multiple MALBAC-amplified single cells to detect reliable nucleotide variants. In contrast to diploid or multiploid cells [29,30,35,59], WGA products of single haploid cells can be used for de novo mutation detection [58]. In these cases WGA nucleotide copy errors can be discriminated from true base variants in the cell, because no heterozygous base variants are expected for unique loci in a haploid cell, offering interesting routes to profiling polar bodies. "
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    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.
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