Coupling mutagenesis and parallel deep sequencing to probe essential residues in a genome or gene

Significance In this work we present a technique called Mut-seq. We show that a very large population of genomes or genes can be mutagenized, selected for growth, and then sequenced to determine which genes or residues are probably essential. Here we have applied this method to T7 bacteriophage and T7-like virus JSF7 of Vibrio cholerae. All essential T7 genes have been previously identified and several DNA replication and transcription proteins have solved structures and are well studied, making this a good model. We use this information to correlate mutability at protein residues with known essentiality, conservation, and predicted structural importance. The sequence of a protein determines its function by influencing its folding, structure, and activity. Similarly, the most conserved residues of orthologous and paralogous proteins likely define those most important. The detection of important or essential residues is not always apparent via sequence alignments because these are limited by the depth of any given gene's phylogeny, as well as specificities that relate to each protein's unique biological origin. Thus, there is a need for robust and comprehensive ways of evaluating the importance of specific amino acid residues of proteins of known or unknown function. Here we describe an approach called Mut-seq, which allows the identification of virtually all of the essential residues present in a whole genome through the application of limited chemical mutagenesis, selection for function, and deep parallel genomic sequencing. Here we have applied this method to T7 bacteriophage and T7-like virus JSF7 of Vibrio cholerae.