Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Abstract

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

“…For eight loci assessed, amplification bias (over-and underrepresentation of sequences) was several orders of magnitude less than for the PCR-based WGA (Dean et al, 2002). Since then, MDA has been applied to many more eukaryotic studies, including the genomic sequencing and genotyping of humans and primates (Lovmar et al, 2003;Barker et al, 2004;Paez et al, 2004;Dickson et al, 2005;Jiang et al, 2005;Lu et al, 2005;Rö nn et al, 2006), insects (Gorrochotegui-Escalante and Black, 2003), fungi (Foster and Monahan, 2005;Gadkar and Rillig, 2005;Fernández-Ortuñ o et al, 2007) and additional applications in forensics and medicine (Hosono et al, 2003;Lasken and Egholm, 2003;Barber and Foran, 2006;Spits et al, 2006;Ballantyne et al, 2007).…”

Allele-specific expression and gene methylation in the control of CYP1A2 mRNA level in human livers

“…Cyclic minisequencing of the PCR products from both gDNA and cDNA, hybridization of the minisequencing reaction products to the captured oligonucleotides (cTags) on the slides and washing of the slides were performed as described previously. 41 Each sample (DNA and the corresponding cDNA) was run in triplicates on the same slide.…”

Successful Genome-Wide Scan in Paired Blood and Buccal Samples

“…Our results have important implications for the conduct of whole genome studies. In studies where buccal samples have been extracted and stored properly, it will be possible to increase sample size and could obviate whole genome amplification before genotyping (11)(12)(13)(14)(15)(16)(17)(18)(19). Although the latter can generate large quantities of DNA from nanograms of highquality DNA, a number of studies have shown that the amplification step is not 100% accurate, and loss of heterozygosity, often clustered in GC-rich loci, can arise (12).…”