Deep indel mutagenesis reveals the impact of insertions and deletions on protein stability and function
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PROVIDER: PRJNA1021220 | ENA |
REPOSITORIES: ENA
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Project description:Amino acid insertions and deletions (indels) are an abundant class of genetic variants. However, compared to substitutions, the effects of indels are not well understood and poorly predicted. Here we address this shortcoming by performing deep indel mutagenesis (DIM) of structurally diverse proteins. Indel tolerance is strikingly different to substitution tolerance and varies extensively both between different proteins and within different regions of the same protein. Although state of the art variant effect predictors perform poorly on indels, we show that both experimentally-measured and computationally-predicted substitution scores can be repurposed as good indel variant effect predictors by incorporating information on protein secondary structures. Quantifying the effects of indels on protein-protein interactions reveals that insertions can be an important class of gain-of-function variants. Our results provide an overview of the impact of indels on proteins and a method to predict their effects genome-wide.
2023-09-28 | GSE244096 | GEO
Project description:We identified 1.96 million small insertions and deletions (INDELs) in the genomes of 79 diverse humans. 10,003 of these INDELs were probed on a custom INDEL genotyping array.
2011-04-19 | GSE27889 | GEO
Project description:Multiplexed assays of variant effects (MAVEs) guide clinical variant interpretation and reveal disease mechanisms. To date, MAVEs have focussed on a single mutation type - amino acid (AA) substitutions - despite the diversity of coding variants that cause disease. Here we use Deep Indel Mutagenesis (DIM) to generate the first comprehensive atlas of diverse variant effects for a disease protein, amyloid beta (Aß) that aggregates in Alzheimer’s disease (AD) and is mutated in familial AD (fAD). The atlas identifies known fAD variants and many mutations beyond substitutions that accelerate Aß aggregation. Truncations, substitutions, insertions, single- and multi-AA deletions differ in their propensity to enhance or impair aggregation, but likely pathogenic variants from all classes are strongly enriched in the polar N-terminus of Aß. This first comparative atlas for any disease gene highlights the importance of including diverse mutation types in MAVEs and provides important mechanistic insights into amyloid nucleation.
2022-10-27 | GSE193837 | GEO
Project description:Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay (Indel-Seq) to monitor rearrangements in human cells at the TRIM37 acceptor locus that reports indels stemming from experimentallyinduced and spontaneous genome instability. Templated insertions, which derive from sequences genome-wide, require contact between donor and acceptor loci, homologous recombination, and are stimulated by DNA end-processing. Insertions are facilitated by transcription and involve a DNA/RNA hybrid intermediate. Indel-Seq reveals that insertions are generated via multiple pathways. The broken acceptor site anneals with a resected DNA break or invades the displaced strand of a transcription bubble or R-loop followed by DNA synthesis, displacement and then ligation by nonhomologous end-joining. Our studies identify transcription-coupled insertions as a critical source of spontaneous genome instability that are distinct from cut-and-paste events
2023-07-03 | GSE234089 | GEO
Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors. Solid tumors in mice were generated by somatic transposon mutagenesis with a PiggyBac transposon system. Insertion sites of transposons in 11 tumors and 6 non-cancerous tail controls were determined by Illumina high-throughput sequencing. Insertions were determined both on 5' and 3' sides of the transposon (PB5 and PB3, respectively). Quantitative analysis of read numbers revealed enrichment of certain insertions in tumors, but not in controls, and these enriched insertions identify candidate cancer genes.
2013-08-28 | E-GEOD-46210 | biostudies-arrayexpress
Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.
2013-08-28 | GSE46210 | GEO
Project description:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs’ endothelial corneal dystrophy (FECD), which can cause blindness through progressive loss of corneal endothelial cells. We established a novel procedure for achieving structural and functional rescue of the post-mitotic corneal endothelium without surgery, using CRISPR/Cas9-based postnatal gene editing in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) at a titer below the cytotoxic threshold, efficiently knocked down COL8A2 protein expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. Here, to determine the indel rate in mouse corneal endothelium, we performed deep sequencing of PCR products (including the target site) amplified from genomic DNA of corneal endothelium. We found that the indel rate was 23.7 ± 4.5% in mouse corneal endothelium. Most insertions were 1bp insertions (19.8 ± 4.0% in total reads), while 2bp deletions were the most frequent (1.0 ± 0.3% in total reads). We moreover found that A or T insertion was predominant, with the proportion of A:T:G:C being 48.7 : 44.6 : 1.8 : 4.9.
2020-09-01 | GSE146998 | GEO
Project description:Insertions and deletions (indels) are common molecular evolutionary events. However, probabilistic models for indel evolution are under-developed due to their computational complexity. Here, we introduce several improvements to indel modeling: 1) While previous models for indel evolution assumed that the rates and length distributions of insertions and deletions are equal, here we propose a richer model that explicitly distinguishes between the two; 2) we introduce numerous summary statistics that allow approximate Bayesian computation-based parameter estimation; 3) we develop a method to correct for biases introduced by alignment programs, when inferring indel parameters from empirical data sets; and 4) using a model-selection scheme, we test whether the richer model better fits biological data compared with the simpler model. Our analyses suggest that both our inference scheme and the model-selection procedure achieve high accuracy on simulated data. We further demonstrate that our proposed richer model better fits a large number of empirical data sets and that, for the majority of these data sets, the deletion rate is higher than the insertion rate.
| S-EPMC8662616 | biostudies-literature
Project description:Modern phylogenetic methods allow inference of ancestral molecular sequences given an alignment and phylogeny relating present-day sequences. This provides insight into the evolutionary history of molecules, helping to understand gene function and to study biological processes such as adaptation and convergent evolution across a variety of applications. Here, we propose a dynamic programming algorithm for fast joint likelihood-based reconstruction of ancestral sequences under the Poisson Indel Process (PIP). Unlike previous approaches, our method, named ARPIP, enables the reconstruction with insertions and deletions based on an explicit indel model. Consequently, inferred indel events have an explicit biological interpretation. Likelihood computation is achieved in linear time with respect to the number of sequences. Our method consists of two steps, namely finding the most probable indel points and reconstructing ancestral sequences. First, we find the most likely indel points and prune the phylogeny to reflect the insertion and deletion events per site. Second, we infer the ancestral states on the pruned subtree in a manner similar to FastML. We applied ARPIP (Ancestral Reconstruction under PIP) on simulated data sets and on real data from the Betacoronavirus genus. ARPIP reconstructs both the indel events and substitutions with a high degree of accuracy. Our method fares well when compared to established state-of-the-art methods such as FastML and PAML. Moreover, the method can be extended to explore both optimal and suboptimal reconstructions, include rate heterogeneity through time and more. We believe it will expand the range of novel applications of ancestral sequence reconstruction. [Ancestral sequences; dynamic programming; evolutionary stochastic process; indel; joint ancestral sequence reconstruction; maximum likelihood; Poisson Indel Process; phylogeny; SARS-CoV.].
| S-EPMC10275563 | biostudies-literature
Project description:Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet compared to missense variants the effects of indels are poorly understood and ineptly predicted. To approach this issue, we developed a sensitive protein folding sensor based on complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor accurately reports on the folding of disease-linked missense variants and artificially designed proteins. Applying the folding sensor to a saturated library of single amino acid indel variants in human DHFR revealed that most regions which tolerate indels are confined to internal loops and the N- and C-termini. Surprisingly, indels are also allowed at a central α-helix. Several indels are temperature-sensitive and the folding of most indels is rescued upon binding to the competitive DHFR inhibitor methotrexate. Predictions using Rosetta and AlphaFold2 correlate with the observed effects, suggesting that most indels operate by destabilizing the native fold and that these computational tools may be useful for classification of indels observed in population sequencing.
2024-07-25 | GSE270811 | GEO