Figure 5

Variant subtraction and filtration. Only a subset of variants in a sample are legitimate candidates that might be responsible for the mutant phenotype of interest. In addition to the ability to map potential mutant lesions to a small region (∼1 Mb), the CloudMap pipeline allows users to subtract nonphenotype-inducing variants from consideration. (A) Subtracting variants present in the background strain. If the premutagenesis, starting strain has been sequenced, users may use the GATK Select Variants tool to subtract starting strain variants (“background variants”) from consideration. (B) Subtracting variants present in other mutant strains from the same screen. If the premutagenesis strain has not been sequenced, then fewer variants can be subtracted from the mutant under consideration. If other mutant strains from the same screen have been sequenced, common variants present in the premutagenesis strain can be deduced from sequence analysis of such mutants. Employing a fairly conservative approach, we can choose to subtract variants only if they are present in only two or more mutants that have been derived from forward genetic screens on the same starting strain. (C) Subtracting variants present in at least one mutant strain of the same background. A less conservative variant subtraction strategy than mentioned in B involves subtracting all variants that are present in the mutant strain of interest and at least one additional strain from the same screen. (D) Subtracting variants present in at least one strain of any background. A more liberal variant subtraction strategy can be performed by subtracting variants present in at least one strain of any background. The same caveats for this strategy apply as for the strategy described above in C. As variant information from more whole genome sequenced strains becomes available, more variants will be available for this subtraction strategy.