The formaldehyde concentration (FA) is indicated. solitary cell DNA by using a competitive spike-in DNA template. Intro Solitary cell genome analysis has become progressively important and offers rapidly developed over the past decade. Two major motivations focus genome analysis on TTP-22 solitary cells. (1) Samples may comprise a very small number of cells or even a solitary cell and there is no choice to use larger samples1,2. (2) Additional samples comprise cells of high genomic variance. Cell heterogeneity takes on a central part in biological phenomena during normal development or disease (e.g., mind development, cancer, or ageing)3C6. In recent years, it has become apparent that cells can acquire genome changes (e.g. mutations, copy number variations (CNV), chromosomal aberrations) that may be propagated to child cells and results in mosaics of cells with different genotypes3,4. Originally caused by a few genomic mutations, multiple changes in solitary cells can result in modified cell programming and cell division rate. To find the clonal development path of mosaic cells, solitary cell genome analysis is a persuasive requirement4,7. To uncover genomic variance in individual cells, methods for deep genome analysis are necessary. These techniques include massively parallel sequencing (known as next generation sequencing, NGS), microarray analysis, or panel real-time PCR analysis. Typically, 1?ng to 1 1?g of DNA is necessary, corresponding to the DNA amount of approximately 102 to 105 human being cells. The DNA amount required for those genome analyses is at least 100-fold higher than the genome content of a single human being cell (6?pg). As a result, accurate amplification of the genomic DNA (whole genome amplification, WGA) is required for reliable genetic analysis. Whole-genome-amplification can generate large amounts from minute quantities of isolated DNA and even from solitary cells8C11. Incomplete or biased genome amplification with missing or underrepresented loci info is a regularly observed limitation when analyzing solitary cell genomes. Besides additional factors, incomplete whole genome amplification is often a result of low template quality12. Genome damage (e.g. DNA breaks, abasic sites, UV induced thymine dimers, formalin revised bases etc.) can occur during cell treatment, harvesting, selection or cell storage. Most of the damaged DNA regions prevent the amplification process at TTP-22 the site of damage. We will refer to these TTP-22 sites as obstructing sites or quit sites. Different methods have been proposed to assess the quality of DNA samples prior to amplification. In the past decade, a couple of quality assays have been developed that address the integrity of DNA. Most of them are based on real-time PCR that quantifies the copy number of in a different way sized PCR products13. However, real-time PCR is limited to small amplicons and performs poorly when measuring DNA integrity over distances larger than 500?bp. Additionally, real-time PCR assays are limited to a small number of genomic loci which may behave in a different way compared to the whole genome. Most important, applying these methods results in the consumption of the solitary cell genome that would not be available for WGA and deep genome analysis. Therefore, none of these methods can be utilized for quality control of a single cell genome. Additional methods use bioinformatic analysis and may be applied only after laborious and cost rigorous microarray or NGS analysis14. We have developed a new method that combines a quality assay of the solitary cell target DNA and whole-genome-amplification (WGA) for further downstream analysis. Here, we present a Control-DNA that is used as competitive spike-in control in solitary cell WGA reactions. The assay makes use of the preferential amplification of long DNA fragments from the Phi29 DNA polymerase. As a result, fragment lengths or distances between polymerase quit sites of Control-DNA and solitary cell DNA are compared during the WGA reaction. The relative amplification rate of Control-DNA after WGA can be ARHGEF11 determined by real-time PCR and inversely correlates with the quality of solitary cell DNA and WGA DNA. Results Mechanism Competitive whole genome amplification (coWGA) is based on multiple displacement amplification (MDA) using the DNA polymerase from phage.