Single Cell Genomics: Advances and Future Perspectives (original) (raw)
Figure 2
Overview of single-cell WGA approaches.
A) Multiple displacement amplification (MDA) initiates with random priming of denatured single-cell DNA template, followed by a 30°C isothermal amplification using a DNA-polymerase with strand-displacement activity, typically phi29 [113]. When the 3′-end of a newly synthesized fragment reaches the 5′-end of an adjoining primed string of nucleotides, it will displace the latter, liberating single-stranded DNA for new primer annealing and DNA-synthesis. B) Primer extension pre-amplification (PEP)-PCR [114], degenerate-oligonucleotide primed (DOP)-PCR [115], and linker-adaptor (LA)-PCR [116] use PCR for WGA. C) WGA methods like PicoPlex [117], [118] and MALBAC [16] use displacement pre-amplification to generate PCR-amplifiable fragments (abbreviated as DA-PCR methods here). Specifically, MALBAC initiates with multiple rounds of displacement pre-amplification using a primer that anneals randomly throughout the genome, but contains a specific sequence allowing full amplicons to form looped pre-amplification products of a cell's template DNA. This looping protects previously copied segments from further pre-amplification. Multiple rounds of the displacement pre-amplification reaction, interspersed by a denaturation step, increase the probability that random priming will occur across the genome. D) Classes of genetic variation reported in single cells following WGA and analysis. 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]. In general, the (DA-)PCR-based WGA products more accurately preserve the copy number profile of the template genome [15]–[17] and can be used for SNP genotyping and base mutation detection [16].