Whole‐Genome Amplification of Single‐Cell Genomes for Next‐Generation Sequencing

Abstract

DNA sequence analysis and genotyping of biological samples using next-generation sequencing (NGS), microarrays, or real-time PCR is often limited by the small amount of sample available. A single cell contains only one to four copies of the genomic DNA, depending on the organism (haploid or diploid organism) and the cell-cycle phase. The DNA content of a single cell ranges from a few femtograms in bacteria to picograms in mammalia. In contrast, a deep analysis of the genome currently requires a few hundred nanograms up to micrograms of genomic DNA for library formation necessary for NGS sequencing or labeling protocols (e.g., microarrays). Consequently, accurate whole-genome amplification (WGA) of single-cell DNA is required for reliable genetic analysis (e.g., NGS) and is particularly important when genomic DNA is limited. The use of single-cell WGA has enabled the analysis of genomic heterogeneity of individual cells (e.g., somatic genomic variation in tumor cells). This unit describes how the genome of single cells can be used for WGA for further genomic studies, such as NGS. Recommendations for isolation of single cells are given and common sources of errors are discussed.

Whole-genome amplification: a useful approach to characterize new genes in unculturable protozoan parasites such asBonamia exitiosa

“…In this sense, the WGA method allows the obtaining of reliable NGS results from limited starting material, reducing also the percentage of undesirable products and enhancing the efficiency of the technique. Some technologies combining both methods have been applied to Eukaryotes and Prokaryotes (Korfhage et al 2013;Pamp et al 2012;Young et al 2012). Data presented in this work demonstrated the successful use of the WGA technique in the unculturable protozoa B. exitiosa which might facilitate the application of new promising methodologies in these organisms.…”

Improved DOP-PCR (iDOP-PCR): A robust and simple WGA method for efficient amplification of low copy number genomic DNA

“…However, these analyses require a small but significant amount of human gDNA, in the range of 1 to 100 ng. This corresponds to 160–16000 human cells [ 1 ] and so these approaches are not appropriate to the analysis of single-cell genomes.…”

“…For samples with limited DNA content, a step of DNA amplification could be used to facilitate further analysis. Whole genome amplification (WGA) is an in vitro method to amplify gDNA and is thus useful in order to obtain sufficient material for analyses of low copy number gDNA (<100 pg), the range typically found when isolating DNA from single cells [ 1 ]. The current WGA techniques involve one of two approaches: isothermal amplification of DNA or thermo-cycling (PCR-based) methods.…”

Improved DOP-PCR (iDOP-PCR): a robust and simple WGA method for efficient amplification of low copy number genomic DNA

“…However, these analyses require a small but significant amount of human gDNA, in the range of 46 1 to 100 ng. This corresponds to 160-16000 human cells [1] and so these approaches are not appropriate to the 47 analysis of single-cell genomes. 48…”

“…Whole genome amplification (WGA) is an in vitro method to amplify gDNA and is thus useful in order 50 to obtain sufficient material for analyses of low copy number gDNA (<100 pg), the range typically found when 51 isolating DNA from single cells [1]. The current WGA techniques involve one of two approaches: isothermal 52 amplification of DNA or thermo-cycling (PCR-based) methods.…”