Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software

Abstract

Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by merely a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features generating and screening libraries for genes sized up to 3600 bases, shotgun sequencing and an enhanced variant-detecting tool. Having processed screens of ∼20 ORF variant libraries, our tool calls variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and its analysis.