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Predicting the Functional Effect of Amino Acid Substitutions and Indels

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      Abstract

      As next-generation sequencing projects generate massive genome-wide sequence variation data, bioinformatics tools are being developed to provide computational predictions on the functional effects of sequence variations and narrow down the search of casual variants for disease phenotypes. Different classes of sequence variations at the nucleotide level are involved in human diseases, including substitutions, insertions, deletions, frameshifts, and non-sense mutations. Frameshifts and non-sense mutations are likely to cause a negative effect on protein function. Existing prediction tools primarily focus on studying the deleterious effects of single amino acid substitutions through examining amino acid conservation at the position of interest among related sequences, an approach that is not directly applicable to insertions or deletions. Here, we introduce a versatile alignment-based score as a new metric to predict the damaging effects of variations not limited to single amino acid substitutions but also in-frame insertions, deletions, and multiple amino acid substitutions. This alignment-based score measures the change in sequence similarity of a query sequence to a protein sequence homolog before and after the introduction of an amino acid variation to the query sequence. Our results showed that the scoring scheme performs well in separating disease-associated variants (n = 21,662) from common polymorphisms (n = 37,022) for UniProt human protein variations, and also in separating deleterious variants (n = 15,179) from neutral variants (n = 17,891) for UniProt non-human protein variations. In our approach, the area under the receiver operating characteristic curve (AUC) for the human and non-human protein variation datasets is ∼0.85. We also observed that the alignment-based score correlates with the deleteriousness of a sequence variation. In summary, we have developed a new algorithm, PROVEAN ( Protein Variation Effect Analyzer), which provides a generalized approach to predict the functional effects of protein sequence variations including single or multiple amino acid substitutions, and in-frame insertions and deletions. The PROVEAN tool is available online at http://provean.jcvi.org.

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      A method and server for predicting damaging missense mutations

      To the Editor: Applications of rapidly advancing sequencing technologies exacerbate the need to interpret individual sequence variants. Sequencing of phenotyped clinical subjects will soon become a method of choice in studies of the genetic causes of Mendelian and complex diseases. New exon capture techniques will direct sequencing efforts towards the most informative and easily interpretable protein-coding fraction of the genome. Thus, the demand for computational predictions of the impact of protein sequence variants will continue to grow. Here we present a new method and the corresponding software tool, PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), which is different from the early tool PolyPhen1 in the set of predictive features, alignment pipeline, and the method of classification (Fig. 1a). PolyPhen-2 uses eight sequence-based and three structure-based predictive features (Supplementary Table 1) which were selected automatically by an iterative greedy algorithm (Supplementary Methods). Majority of these features involve comparison of a property of the wild-type (ancestral, normal) allele and the corresponding property of the mutant (derived, disease-causing) allele, which together define an amino acid replacement. Most informative features characterize how well the two human alleles fit into the pattern of amino acid replacements within the multiple sequence alignment of homologous proteins, how distant the protein harboring the first deviation from the human wild-type allele is from the human protein, and whether the mutant allele originated at a hypermutable site2. The alignment pipeline selects the set of homologous sequences for the analysis using a clustering algorithm and then constructs and refines their multiple alignment (Supplementary Fig. 1). The functional significance of an allele replacement is predicted from its individual features (Supplementary Figs. 2–4) by Naïve Bayes classifier (Supplementary Methods). We used two pairs of datasets to train and test PolyPhen-2. We compiled the first pair, HumDiv, from all 3,155 damaging alleles with known effects on the molecular function causing human Mendelian diseases, present in the UniProt database, together with 6,321 differences between human proteins and their closely related mammalian homologs, assumed to be non-damaging (Supplementary Methods). The second pair, HumVar3, consists of all the 13,032 human disease-causing mutations from UniProt, together with 8,946 human nsSNPs without annotated involvement in disease, which were treated as non-damaging. We found that PolyPhen-2 performance, as presented by its receiver operating characteristic curves, was consistently superior compared to PolyPhen (Fig. 1b) and it also compared favorably with the three other popular prediction tools4–6 (Fig. 1c). For a false positive rate of 20%, PolyPhen-2 achieves the rate of true positive predictions of 92% and 73% on HumDiv and HumVar, respectively (Supplementary Table 2). One reason for a lower accuracy of predictions on HumVar is that nsSNPs assumed to be non-damaging in HumVar contain a sizable fraction of mildly deleterious alleles. In contrast, most of amino acid replacements assumed non-damaging in HumDiv must be close to selective neutrality. Because alleles that are even mildly but unconditionally deleterious cannot be fixed in the evolving lineage, no method based on comparative sequence analysis is ideal for discriminating between drastically and mildly deleterious mutations, which are assigned to the opposite categories in HumVar. Another reason is that HumDiv uses an extra criterion to avoid possible erroneous annotations of damaging mutations. For a mutation, PolyPhen-2 calculates Naïve Bayes posterior probability that this mutation is damaging and reports estimates of false positive (the chance that the mutation is classified as damaging when it is in fact non-damaging) and true positive (the chance that the mutation is classified as damaging when it is indeed damaging) rates. A mutation is also appraised qualitatively, as benign, possibly damaging, or probably damaging (Supplementary Methods). The user can choose between HumDiv- and HumVar-trained PolyPhen-2. Diagnostics of Mendelian diseases requires distinguishing mutations with drastic effects from all the remaining human variation, including abundant mildly deleterious alleles. Thus, HumVar-trained PolyPhen-2 should be used for this task. In contrast, HumDiv-trained PolyPhen-2 should be used for evaluating rare alleles at loci potentially involved in complex phenotypes, dense mapping of regions identified by genome-wide association studies, and analysis of natural selection from sequence data, where even mildly deleterious alleles must be treated as damaging. Supplementary Material 1
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        A map of human genome variation from population-scale sequencing.

        The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data set. On average, each person is found to carry approximately 250 to 300 loss-of-function variants in annotated genes and 50 to 100 variants previously implicated in inherited disorders. We demonstrate how these results can be used to inform association and functional studies. From the two trios, we directly estimate the rate of de novo germline base substitution mutations to be approximately 10(-8) per base pair per generation. We explore the data with regard to signatures of natural selection, and identify a marked reduction of genetic variation in the neighbourhood of genes, due to selection at linked sites. These methods and public data will support the next phase of human genetic research.
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          Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences.

          In 2001 and 2002, we published two papers (Bioinformatics, 17, 282-283, Bioinformatics, 18, 77-82) describing an ultrafast protein sequence clustering program called cd-hit. This program can efficiently cluster a huge protein database with millions of sequences. However, the applications of the underlying algorithm are not limited to only protein sequences clustering, here we present several new programs using the same algorithm including cd-hit-2d, cd-hit-est and cd-hit-est-2d. Cd-hit-2d compares two protein datasets and reports similar matches between them; cd-hit-est clusters a DNA/RNA sequence database and cd-hit-est-2d compares two nucleotide datasets. All these programs can handle huge datasets with millions of sequences and can be hundreds of times faster than methods based on the popular sequence comparison and database search tools, such as BLAST.
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            Author and article information

            Affiliations
            The J. Craig Venter Institute, Rockville, Maryland, United States of America
            UMR-S665, INSERM, Université Paris Diderot, INTS, France
            Author notes

            Competing Interests: The authors have the following competing interests: The authors have developed a new algorithm, PROVEAN (Protein Variation Effect Analyzer), which provides a generalized approach to predict the functional effects of protein sequence variations including single or multiple amino acid substitutions, and in-frame insertions and deletions. The PROVEAN tool is available online at http://provean.jcvi.org. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

            Conceived and designed the experiments: APC YC GES. Performed the experiments: YC. Analyzed the data: YC GES APC JRM SM. Wrote the paper: APC YC.

            [¤a]

            Current address: Department of Bioinformatics, Pathway Genomics Corporation, San Diego, California, United States of America

            [¤b]

            Current address: Howard Hughes Medical Institute Janelia Farm Research Campus, Ashburn, Virginia, United States of America

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            1932-6203
            2012
            8 October 2012
            : 7
            : 10
            3466303
            23056405
            PONE-D-12-10334
            10.1371/journal.pone.0046688
            (Editor)

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Counts
            Pages: 13
            Funding
            The work described is funded by the National Institutes of Health (grant number 5R01HG004701-03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology
            Biochemistry
            Proteins
            Protein Structure
            Computational Biology
            Macromolecular Structure Analysis
            Protein Structure
            Population Genetics
            Mutation
            Sequence Analysis
            Genetics
            Genetic Mutation
            Mutation Types
            Genomics
            Genome Analysis Tools
            Genomic Medicine

            Uncategorized

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