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. 2017 Aug 1;45(15):9206–9217. doi: 10.1093/nar/gkx691

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© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Effect of polymerase on assembly quality. We assembled two different 220 bp constructs (C1 and C2) from five 60 nt oligos with 20 bp overlaps with Q5 and KAPA2G Robust polymerases. (A) We find that the average error frequency of both constructs is significantly higher for KAPA2G Robust than for Q5 (9.7 versus 2.5 errors/kb). We observe similar trends for the average percentage of perfect assemblies (60.5% for Q5 and 10.4% for KAPA2G Robust). (B) Similar to the two-oligo assembly, we find that the Taq-based KAPA2G Robust polymerase also has a higher rate of transitions than transversions (mean of versus over both constructs; Mann–Whitney U, P << 0.001). (C) We find that the median rate of multiple base deletions per base in the overlap regions decreased ∼2-fold relative to non-overlapping regions for both polymerases (Mann–Whitney U, P << 0.001). Similarly, the median rate of multiple base deletions per base also significantly decreases in the priming regions for both KAPA2G Robust(∼6-fold) and Q5 (∼13-fold) for both constructs (both Mann–Whitney U, P << 0.001). The difference in decrease between the polymerases was not significant.

Inline graphic — Effect of polymerase on assembly quality. We assembled two different 220 bp constructs (C1 and C2) from five 60 nt oligos with 20 bp overlaps with Q5 and KAPA2G Robust polymerases. (A) We find that the average error frequency of both constructs is significantly higher for KAPA2G Robust than for Q5 (9.7 versus 2.5 errors/kb). We observe similar trends for the average percentage of perfect assemblies (60.5% for Q5 and 10.4% for KAPA2G Robust). (B) Similar to the two-oligo assembly, we find that the Taq-based KAPA2G Robust polymerase also has a higher rate of transitions than transversions (mean of versus over both constructs; Mann–Whitney U, P << 0.001). (C) We find that the median rate of multiple base deletions per base in the overlap regions decreased ∼2-fold relative to non-overlapping regions for both polymerases (Mann–Whitney U, P << 0.001). Similarly, the median rate of multiple base deletions per base also significantly decreases in the priming regions for both KAPA2G Robust(∼6-fold) and Q5 (∼13-fold) for both constructs (both Mann–Whitney U, P << 0.001). The difference in decrease between the polymerases was not significant.