Site-directed mutagenesis can overcome uncertainty caused by the possibility of false negatives. In our previous work [8] , we introduced all of the mutations identified into the wild type strain and were able to reconstruct the observed change in growth phenotype in 4 of 5 cases, indicating that all of the important mutations were found in most cases. An additional precaution against false negatives is resequencing multiple replicates from the same experimental setup. Our previous work identified mutations in the gene glpK in 4 of the 5 replicates. Sequencing glpK from the one remaining replicate with other technology showed a 9 bp duplication undetected by CGS. This demonstrates the value of replicates and why genes containing mutations in some replicates should be screened for false negatives in the other replicates.