DnaG variants improve CoS-MAGE Performance. EcNR2, EcNR2.dnaG.K580A, EcNR2.dnaG.Q576A and Nuc5-.dnaG.Q576A were tested for their performance in CoS-MAGE using 3 sets of 10 oligos as described in B. For each set, all 10 alleles were simultaneously assayed by mascPCR in recombinant clones after 1 cycle of CoS-MAGE. (**A**) The data are presented using stacked AR frequency plots, which show the distribution of clones exhibiting a given number of allele conversions. (**B**) Mean number of alleles converted for each strain are shown with *P*-values indicated above the bars. Statistical significance is denoted using a star system where * denotes *P* < 0.003, ** denotes *P* < 0.001 and *** denotes *P* < 0.0001. The data are presented as the mean (reported numerically inside each bar) ± SEM. (**C**) AR frequencies for each individual allele are shown for all tested strains. Overall, the relative performance of each strain was Nuc5-.dnaG.Q576A > EcNR2.dnaG.Q576A > EcNR2.dnaG.K580A > EcNR2. This trend reflects an improvement commensurate with the severity of primase attenuation (i.e. the Q576A variant has more severely disrupted primase and larger OFs than the K580A variant). Furthermore, Nuc5-.dnaG.Q576A combines the benefits of the DnaG Q576A variant and the benefits of the inactivation of five potent exonucleases (Mosberg, J.A., Gregg, C.J., *et al.*, in review). For Set 1: EcNR2, *n* = 319; EcNR2.dnaG.K580A, *n* = 93; EcNR2.dnaG.Q576A, *n* = 141; Nuc5^{-}.dnaG.Q576A, *n* = 47. For Set 2: EcNR2, *n* = 269; EcNR2.dnaG.K580A, *n* = 111; EcNR2.dnaG.Q576A, *n* = 236; Nuc5^{-}.dnaG.Q576A, *n* = 191. For set 3: EcNR2, *n* = 327; EcNR2.dnaG.K580A, *n* = 136; EcNR2.dnaG.Q576A, *n* = 184; Nuc5^{-}.dnaG.Q576A, *n* = 92.

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