A mechanistic basis for amplification differences between samples and between genome regions

Abstract

BackgroundFor many analytical methods the efficiency of DNA amplification varies across the genome and between samples. The most affected genome regions tend to correlate with high C + G content, however this relationship is complex and does not explain why the direction and magnitude of effects varies considerably between samples.ResultsHere, we provide evidence that sequence elements that are particularly high in C + G content can remain annealed even when aggressive melting conditions are applied. In turn, this behavior creates broader ‘Thermodynamically Ultra-Fastened’ (TUF) regions characterized by incomplete denaturation of the two DNA strands, so reducing amplification efficiency throughout these domains.ConclusionsThis model provides a mechanistic explanation for why some genome regions are particularly difficult to amplify and assay in many procedures, and importantly it also explains inter-sample variability of this behavior. That is, DNA samples of varying quality will carry more or fewer nicks and breaks, and hence their intact TUF regions will have different lengths and so be differentially affected by this amplification suppression mechanism – with ‘higher’ quality DNAs being the most vulnerable. A major practical consequence of this is that inter-region and inter-sample variability can be largely overcome by employing routine fragmentation methods (e.g. sonication or restriction enzyme digestion) prior to sample amplification.

Post-zygotic Point Mutations Are an Underrecognized Source of De Novo Genomic Variation

“…A limitation of both of these techniques is that they might show an allelic imbalance as a result of biased amplification of one allele over the other. 28 For the most part, significant deviations in the allelic ratio secondary to technical artifacts observed in Sanger sequencing and ADS were method-specific rather than reproducible PCR artifacts ( Figure S12). We have attempted to remedy this problem by using smMIPs, which provide targeted high sequence coverage and the ability to identify individual captured molecules 23 and thus prevent any allelic-ratio deviations resulting from PCR amplification bias.…”

Clinical Application of NGS Tools in the Diagnosis of Collagenopathies

“…CG-rich regions, such as CpG islands, could decrease the depth of coverage because these regions denature, causing difficulties during amplification. 8 It is important for the success of the experiment and for a correct variant analysis to maintain the uniformity of coverage. Nevertheless, custom target sequencing is the best method if the genes clinically related to disease are known.…”

Multi-template polymerase chain reaction

“…Amplification efficiency can vary across a genome [84], [85]. Genomic regions resistant to amplification by PCR correlate with regions having a high GC content as these do not denature efficiently under routinely used conditions [64], [86].…”

“…Genomic regions resistant to amplification by PCR correlate with regions having a high GC content as these do not denature efficiently under routinely used conditions [64], [86]. Veal and co-workers [85] proposed that regions of extreme GC content remain duplexed during standard DNA denaturation procedures, and in so doing also prevented their flanking regions from separating. As such, these neighboring strands are able to quickly reanneal as soon as non-denaturing conditions are re-established.…”