Structural alterations from multiple displacement amplification of a human genome revealed by mate-pair sequencing

Abstract

Comprehensive identification of the acquired mutations that cause common cancers will require genomic analyses of large sets of tumor samples. Typically, the tissue material available from tumor specimens is limited, which creates a demand for accurate template amplification. We therefore evaluated whether phi29-mediated whole genome amplification introduces false positive structural mutations by massive mate-pair sequencing of a normal human genome before and after such amplification. Multiple displacement amplification led to a decrease in clone coverage and an increase by two orders of magnitude in the prevalence of inversions, but did not increase the prevalence of translocations. While multiple strand displacement amplification may find uses in translocation analyses, it is likely that alternative amplification strategies need to be developed to meet the demands of cancer genomics.

Figure 1. Introduction of coverage bias by multiple displacement amplification.

A. Fold base coverage per bin of 10 MBp consecutive bases before (blue line) and after (red line) whole genome amplification. B. Logarithmic quantile-quantile plot of the binned distributions (bin size 1000 nt) of the sum of the coverage of both tags of a mate-pair.

Figure 2. Multiple strand displacement of a normal human genome introduces inversions.

Inversions (red bars) supported by at least 4 independent mate-pairs after shotgun genome sequencing of a non-amplified (A) and MDA amplified (B) genome.

Figure 3. Loss of sensitivity in breakpoint detection by multiple strand displacement-induced inversions.

Forward (blue) and reverse (red) tags in mate-pairs (grey line) surrounding the start and end breakpoints of the inversion chr16∶83744887–83748786 along with 5 kb flanking regions on chromosome 16 (black line). The tags mapping to the forward chromosome strand are plotted above the chromosome line, and the tags mapping to the reverse strand are plotted below. The inversion start and end regions identified are shown as pink and yellow bands, respectively.