Measuring the activity of protein variants on a large scale using deep mutational scanning

Abstract

Deep mutational scanning marries selection for protein function to high-throughput DNA sequencing in order to quantify the activity of variants of a protein on a massive scale. First, an appropriate selection system for the protein function of interest is identified and validated. Second, a library of variants is created, introduced into the selection system and subjected to selection. Third, library DNA is recovered throughout the selection and deeply sequenced. Finally, a functional score for each variant is calculated based on the change in the frequency of the variant during the selection. This protocol describes the steps that must be carried out to generate a large-scale mutagenesis data set consisting of functional scores for up to hundreds of thousands of variants of a protein of interest. Establishing an assay, generating a library of variants, and carrying out a selection and its accompanying sequencing takes on the order of 4–6 weeks; the initial data analysis can be completed in 1 week.

An extended set of yeast-based functional assays accurately identifies human disease mutations

“…Modifier screens in model organisms offer a powerful means not only for understanding disease mechanism and identifying additional disease genes, but also for identifying potential therapeutics (Tardiff et al 2014). The experimental throughput of model organisms offers the opportunity to build rich genotypephenotype maps through saturation mutagenesis and deep sequencing-see, for example, Fowler et al (2014) and Kitzman et al (2015)-potentially providing a 'look-up table' for functionality of human variants before they have ever been observed in the clinic.…”

An Engineered Switch in T Cell Receptor Specificity Leads to an Unusual but Functional Binding Geometry

“…Sorted libraries had 4.2×10 5 – 1.9×10 6 reads. Enrichment ratios, E x i = log 2 ( f x i [sorted] / f x i [naïve]) where x is the amino acid identity, i is the position and f is the sequence frequency, were calculated using modified scripts from Enrich software (Fowler et al, 2014). …”

Variant Interpretation: Functional Assays to the Rescue

“…Some aspects of MAVEs, namely library synthesis and sequencing, are relatively well established and have been described extensively. 12,13,[31][32][33] Here, we focus on the aspects that are critical for the goal of prospectively interpreting human genetic variation.…”