A deep mutational scanning experiment.(A) A gene is mutagenized to create a library that contains all single codon mutations. The mutant library is introduced into cells or viruses and subjected to a functional selection that enriches beneficial mutations and depletes deleterious ones. Deep sequencing is used to count mutations in a sample of the variants present pre- and post-selection. Using dms_tools, the data can be analyzed to infer the “preference” of each site for each amino acid; in the visualization, letter heights are proportional to the preference for that amino acid. (B) The experiment can be extended by subjecting the library of functional variants to two different selection pressures, and using deep sequencing to assess which variants are favored in one condition versus the other. Using dms_tools, the data can be analyzed to infer the “differential preference” of each site for each amino acid in the alternative selection s2 versus the control selection s1; in the visualization, letter heights above or below the line are proportional to the differential preference for or against that amino acid.