Genome coverage and sequence fidelity of  29 polymerase-based multiple strand displacement whole genome amplification

Abstract

Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of phi29 polymerase-based genome amplification (phi29MDA) using direct sequencing and high density oligonucleotide arrays probing >10,000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500,000 bp, the estimated error rate (9.5 x 10(-6)) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that phi29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.

Whole-genome sequence of Schistosoma haematobium

“…After assembly, all usable reads were realigned to scaffolds to assess single-base accuracy of the assembled genome sequence. We did not find evidence of GC-biased nonrandom sampling 10 or of artifacts induced by multiple displacement amplification, a result that is consistent with published information 11,12 (Supplementary Note, Supplementary Figs. 3 and 4).…”

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).…”

Rolling circle amplification of metazoan mitochondrial genomes

“…Once the primers have annealed to the DNA, the phi29 DNA polymerase extends the annealed primers and, because of its strong strand displacement activity and high processivity, generates long concatenamers of the circular template DNA in a single isothermal reaction. The phi29 polymerase also has an inherent 3Ј-5Ј proofreading exonculease activity, accounting for its high Wdelity (Esteban et al, 1993;Paez et al, 2004). Despite the name, this reaction does not work only on circular DNA; linear DNA also will undergo repeated rounds of simple strand displacement ampliWcation (Lizardi et al, 1998), so it is critical that the DNA preparation be highly enriched for mtDNA, although the amount can be minuscule.…”