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This Perspective discusses the power of large mutational scans for the study of protein properties, the analytical challenges posed by the resulting data sets and the potential of this approach to further our understanding of human genetic variation. Mutagenesis provides insight into proteins, but only recently have assays that couple genotype to phenotype been used to assess the activities of as many as 1 million mutant versions of a protein in a single experiment. This approach—'deep mutational scanning'—yields large-scale data sets that can reveal intrinsic protein properties, protein behavior within cells and the consequences of human genetic variation. Deep mutational scanning is transforming the study of proteins, but many challenges must be tackled for it to fulfill its promise.

Multiplex assays of variant effect (MAVEs), such as deep mutational scans and massively parallel reporter assays, test thousands of sequence variants in a single experiment. Despite the importance of MAVE data for basic and clinical research, there is no standard resource for their discovery and distribution. Here, we present MaveDB ( https://www.mavedb.org ), a public repository for large-scale measurements of sequence variant impact, designed for interoperability with applications to interpret these datasets. We also describe the first such application, MaveVis, which retrieves, visualizes, and contextualizes variant effect maps. Together, the database and applications will empower the community to mine these powerful datasets.

Mutagenesis is a widely used method for identifying protein positions that are important for function or ligand binding. Advances in high-throughput DNA sequencing and mutagenesis techniques have enabled measurement of the effects of nearly all possible amino acid substitutions in many proteins. The resulting large-scale mutagenesis data sets offer a unique opportunity to draw general conclusions about the effects of different amino acid substitutions. Thus, we analyzed 34,373 mutations in 14 proteins whose effects were measured using large-scale mutagenesis approaches. Methionine was the most tolerated substitution, while proline was the least tolerated. We found that several substitutions, including histidine and asparagine, best recapitulated the effects of other substitutions, even when the identity of the wild-type amino acid was considered. The effects of histidine and asparagine substitutions also correlated best with the effects of other substitutions in different structural contexts. Furthermore, highly disruptive substitutions like aspartic and glutamic acid had the most discriminatory power for detecting ligand interface positions. Our work highlights the utility of large-scale mutagenesis data, and our conclusions can help guide future single substitution mutational scans.

Deep mutational scanning is a widely used method for multiplex measurement of functional consequences of protein variants. We developed a new deep mutational scanning statistical model that generates error estimates for each measurement, capturing both sampling error and consistency between replicates. We apply our model to one novel and five published datasets comprising 243,732 variants and demonstrate its superiority in removing noisy variants and conducting hypothesis testing. Simulations show our model applies to scans based on cell growth or binding and handles common experimental errors. We implemented our model in Enrich2, software that can empower researchers analyzing deep mutational scanning data.

In this protocol, the authors describe deep mutational scanning, an approach that involves selecting for protein function followed by high-throughput DNA sequencing and enables quantification of the activity of protein variants on a massive scale. 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 deep-sequenced. Finally, a functional score for each variant is calculated on the basis of 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.

Analysis of protein mutants is an effective means to understand their function. Protein display is an approach that allows large numbers of mutants of a protein to be selected based on their activity, but only a handful with maximal activity have been traditionally identified for subsequent functional analysis. However, the recent application of high-throughput sequencing (HTS) to protein display and selection has enabled simultaneous assessment of the function of hundreds of thousands of mutants that span the activity range from high to low. Such deep mutational scanning approaches are rapid and inexpensive with the potential for broad utility. In this review, we discuss the emergence of deep mutational scanning, the challenges associated with its use and some of its exciting applications.

Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing.

The ability of a protein to carry out a given function results from fundamental physicochemical properties that include the protein’s structure, mechanism of action, and thermodynamic stability. Traditional approaches to study these properties have typically required the direct measurement of the property of interest, oftentimes a laborious undertaking. Although protein properties can be probed by mutagenesis, this approach has been limited by its low throughput. Recent technological developments have enabled the rapid quantification of a protein’s function, such as binding to a ligand, for numerous variants of that protein. Here, we measure the ability of 47,000 variants of a WW domain to bind to a peptide ligand and use these functional measurements to identify stabilizing mutations without directly assaying stability. Our approach is rooted in the well-established concept that protein function is closely related to stability. Protein function is generally reduced by destabilizing mutations, but this decrease can be rescued by stabilizing mutations. Based on this observation, we introduce partner potentiation, a metric that uses this rescue ability to identify stabilizing mutations, and identify 15 candidate stabilizing mutations in the WW domain. We tested six candidates by thermal denaturation and found two highly stabilizing mutations, one more stabilizing than any previously known mutation. Thus, physicochemical properties such as stability are latent within these large-scale protein functional data and can be revealed by systematic analysis. This approach should allow other protein properties to be discovered.

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here, we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin‐like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes. Synopsis A new framework combining random codon‐mutagenesis and multiplexed functional variation assays with computational imputation, allows the comprehensive identification of functional missense variation. The approach is applied to identify pathogenic variation in six human genes. A modular deep mutational scanning (DMS) framework combines random codon‐mutagenesis and multiplexed functional variation assays with computational imputation and refinement. The framework is applied to four human proteins corresponding to six human genes and generates comprehensive functional variation maps covering > 13,000 missense variants. These maps confidently identify pathogenic variation. DMS is a promising approach for generating exhaustive maps of functional variation covering all human genes. Graphical Abstract A new framework combining random codon‐mutagenesis and multiplexed functional variation assays with computational imputation, allows the comprehensive identification of functional missense variation. The approach is applied to identify pathogenic variation in six human genes.

The combination of protein display, moderate selection for protein activity and high-throughput DNA sequencing can be applied to hundreds of thousands of protein variants in parallel, enabling the derivation of sequence-function relationships. We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.