Genome amplification of single sperm using multiple displacement amplification

Department of Environmental Health, Center for Genome Information, University of Cincinnati College of Medicine 3223 Eden Ave, Cincinnati, OH 45267, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2005; 33(10):e91. DOI: 10.1093/nar/gni089
Source: PubMed


Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 mul reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.

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    • "[14]–[16] However, the extremely low concentration of DNA found in a single cell genome in the still large volume of the WGA mixture (20–50 µl) often gives rise to non-specific amplification and amplification biases. [17]–[19] In addition, sorting and manipulation of individual cells to perform single cell analysis can be very challenging and each manipulation can give rise to loss of material. Fluorescence activated cell sorting (FACS) [8], [20], micromanipulation [10], [11], laser capture microdissection (LCM) [9], [21] and DEP Array [22], [23] have all been applied for single cell isolation, but processing of the cells to obtain DNA for downstream analysis (e.g. "
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    ABSTRACT: The heterogeneity of tumor cells and their alteration during the course of the disease urges the need for real time characterization of individual tumor cells to improve the assessment of treatment options. New generations of therapies are frequently associated with specific genetic alterations driving the need to determine the genetic makeup of tumor cells. Here, we present a microfluidic device for parallel single cell whole genome amplification (pscWGA) to obtain enough copies of a single cell genome to probe for the presence of treatment targets and the frequency of its occurrence among the tumor cells. Individual cells were first captured and loaded into eight parallel amplification units. Next, cells were lysed on a chip and their DNA amplified through successive introduction of dedicated reagents while mixing actively with the help of integrated button-valves. The reaction chamber volume for scWGA 23.85 nl, and starting from 6-7 pg DNA contained in a single cell, around 8 ng of DNA was obtained after WGA, representing over 1000-fold amplification. The amplified products from individual breast cancer cells were collected from the device to either directly investigate the amplification of specific genes by qPCR or for re-amplification of the DNA to obtain sufficient material for whole genome sequencing. Our pscWGA device provides sufficient DNA from individual cells for their genetic characterization, and will undoubtedly allow for automated sample preparation for single cancer cell genomic characterization.
    PLoS ONE 09/2014; 9(9):e107958. DOI:10.1371/journal.pone.0107958 · 3.23 Impact Factor
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    • "The commercially available MDA reagents have frequently been reported as being contaminated by unwanted DNA. Several authors dealing with MDA contamination concluded that contamination did not come from human DNA or other target genomes, but could originate from hexamer concatenation or from the enzyme preparation process, including host bacteria Bacillus subtilis [18], [20], [31], [39]–[41]. Theoretically, samples from the same genomic origin should present equivalent or similar hexamer content. This method was previously used to compare metagenomic sources, allowing the discrimination of samples with potential contamination [42]. "
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    ABSTRACT: The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli fs 24 from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA.
    PLoS ONE 06/2014; 9(6):e97379. DOI:10.1371/journal.pone.0097379 · 3.23 Impact Factor
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    • "PCR-based methodology has been used frequently to study haplotypes and recombination events in single sperm cells e.g. (Hubert et al. 1994), and MDA has enabled extensive genotyping (Jiang et al. 2005). Recently, genome-wide analysis of individual sperm cells after MDA was used to assess recombination activity and de novo mutation rates (Wang et al. 2012). "
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    ABSTRACT: There is increasing evidence that the phenotypic effects of genomic sequence variants are best understood in terms of variant haplotypes rather than as isolated polymorphisms. Haplotype analysis is also critically important for uncovering population histories, and for the study of evolutionary genetics. Although the sequencing of individual human genomes to reveal personal collections of sequence variants is now well established, there has been slower progress in the phasing of these variants into pairs of haplotypes along each pair of chromosomes. Here, we have developed a distinct approach to haplotyping that can yield chromosome-length haplotypes, including the vast majority of heterozygous SNPs in an individual human genome. This approach exploits the haploid nature of sperm cells, and employs a combination of genotyping and low-coverage sequencing on a short-read platform. In addition to generating chromosome-length haplotypes, the approach can directly identify recombination events (averaging 1.1 per chromosome) with a median resolution of less than 100 kb.
    Genome Research 01/2013; 23(5). DOI:10.1101/gr.144600.112 · 14.63 Impact Factor
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