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Termites (Insecta, Blattodea, Termitoidae) are a widespread and diverse group of eusocial insects known for their ability to digest wood matter. Herein, we report the draft genome of the subterranean termite Reticulitermes lucifugus, an economically important species and among the most studied taxa with respect to eusocial organization and mating system. The final assembly (~813 Mb) covered up to 88% of the estimated genome size and, in agreement with the Asexual Queen Succession Mating System, it was found completely homozygous. We predicted 16,349 highly supported gene models and 42% of repetitive DNA content. Transposable elements of R. lucifugus show similar evolutionary dynamics compared to that of other termites, with two main peaks of activity localized at 25% and 8% of Kimura divergence driven by DNA, LINE and SINE elements. Gene family turnover analyses identified multiple instances of gene duplication associated with R. lucifugus diversification, with significant lineage-specific gene family expansions related to development, perception and nutrient metabolism pathways. Finally, we analysed P450 and odourant receptor gene repertoires in detail, highlighting the large diversity and dynamical evolutionary history of these proteins in the R. lucifugus genome. This newly assembled genome will provide a valuable resource for further understanding the molecular basis of termites biology as well as for pest control.

Genetics play a key role in venous thromboembolism (VTE) risk, however established risk factors in European populations do not translate to individuals of African descent because of the differences in allele frequencies between populations. As part of the fifth iteration of the Critical Assessment of Genome Interpretation, participants were asked to predict VTE status in exome data from African American subjects. Participants were provided with 103 unlabeled exomes from patients treated with warfarin for non-VTE causes or VTE and asked to predict which disease each subject had been treated for. Given the lack of training data, many participants opted to use unsupervised machine learning methods, clustering the exomes by variation in genes known to be associated with VTE. The best performing method using only VTE related genes achieved an area under the ROC curve of 0.65. Here, we discuss the range of methods used in the prediction of VTE from sequence data and explore some of the difficulties of conducting a challenge with known confounders. In addition, we show that an existing genetic risk score for VTE that was developed in European subjects works well in African Americans.

This paper reports the evaluation of predictions for the ``CALM1'' challenge in the fifth round of the Critical Assessment of Genome Interpretation held in 2018. In the challenge, the participants were asked to predict effects on yeast growth caused by missense variants of human calmodulin, a highly conserved protein in eukaryotic cells sensing calcium concentration. The performance of predictors implementing different algorithms and methods is similar. Most predictors are able to identify the deleterious or tolerated variants with modest accuracy, with a baseline predictor based purely on sequence conservation slightly outperforming the submitted predictions. Nevertheless, we think that the accuracy of predictions remains far from satisfactory, and the field awaits substantial improvements. The most poorly predicted variants in this round surround functional CALM1 sites that bind calcium or peptide, which suggests that better incorporation of structural analysis may help improve predictions.

Precision medicine and sequence-based clinical diagnostics seek to predict disease risk or to identify causative variants from sequencing data. The Critical Assessment of Genome Interpretation (CAGI) is a community experiment consisting of genotype-phenotype prediction challenges; participants build models, undergo assessment, and share key findings. In the past, few CAGI challenges have addressed the impact of sequence variants on splicing. In CAGI5, two challenges (Vex-seq and MaPSY) involved prediction of the effect of variants, primarily single-nucleotide changes, on splicing. Although there are significant differences between these two challenges, both involved prediction of results from high-throughput exon inclusion assays. Here, we discuss the methods used to predict the impact of these variants on splicing, their performance, strengths, and weaknesses, and prospects for predicting the impact of sequence variation on splicing and disease phenotypes.

The Bologna ENZyme Web Server (BENZ WS) annotates four-level Enzyme Commission numbers (EC numbers) as defined by the International Union of Biochemistry and Molecular Biology (IUBMB). BENZ WS filters a target sequence with a combined system of Hidden Markov Models, modelling protein sequences annotated with the same molecular function, and Pfams, carrying along conserved protein domains. BENZ returns, when successful, for any enzyme target sequence an associated four-level EC number. Our system can annotate both monofunctional and polyfunctional enzymes, and it can be a valuable resource for sequence functional annotation., Graphical Abstract Graphical AbstractBENZ WS annotates with a four-digit EC number a query protein. Its association with EC code/s depends on the interplay of the two different sets of Hidden Markov Models (HMMs), considering protein domains, and clusters of similar sequences.

Protein-solvent interaction provides important features for protein surface engineering when the structure is absent or partially solved. Presently, we can integrate the notion of solvent exposed/buried residues with that of their flexibility and intrinsic disorder to highlight regions where mutations may increase or decrease protein stability in order to modify proteins for biotechnological reasons, while preserving their functional integrity. Here we describe a web server, which provides the unique possibility of integrating knowledge of solvent and non-solvent exposure with that of residue conservation, flexibility and disorder of a protein sequence, for a better understanding of which regions are relevant for protein integrity. The core of the webserver is DeepREx, a novel deep learning-based tool that classifies each residue in the sequence as buried or exposed. DeepREx is trained on a high-quality, non-redundant dataset derived from the Protein Data Bank comprising 2332 monomeric protein chains and benchmarked on a blind test set including 200 protein sequences unrelated with the training set. Results show that DeepREx performs at the state-of-the-art in the field. In turn, the Web Server, DeepREx-WS, supplements the predictions of DeepREx with features that allow a better characterisation of exposed and buried regions: i) residue conservation derived from multiple sequence alignment; ii) local sequence hydrophobicity; iii) residue flexibility computed with MEDUSA; iv) a predictor of secondary structure; v) the presence of disordered regions as derived from MobiDB-Lite3.0. The web server allows browsing, selecting and intersecting the different features. We demonstrate a possible application of the DeepREx-WS for assisting the identification of residues to be variated in protein surface engineering processes.

Crustaceans of the order Notostraca (Branchiopoda) are distributed worldwide and are known for the remarkable morphological stasis between their extant and Permian fossil species. Moreover, these crustaceans show relevant ecological traits and a wide range of reproductive strategies. However, genomic studies on notostracans are fairly limited. Here, we present the genome sequences of two notostracan taxa, Lepidurus arcticus and Lepidurus apus lubbocki. Taking advantage of the small genome sizes (~0.11pg) of these taxa, genomes were sequenced for one individual per species with one run on the Illumina HiSeq X platform. We finally assembled 73.2Mbp (L.arcticus) and 90.3Mbp (L.apus lubbocki) long genomes. Assemblies cover up to 84% of the estimated genome size, with a gene completeness >97% for both genomes. In total, 13%–16% of the assembled genomes consist of repeats, and based on read mapping, L.apus lubbocki shows a significantly lower transposable element content than L.arcticus. The analysis of 2,376 orthologous genes indicates an ~7% divergence between the two Lepidurus taxa, with a nucleotide substitution rate significantly lower than that of Daphnia taxa. Ka/Ks analysis suggests purifying selection in both branchiopod lineages, raising the question of whether the low substitution rate of Lepidurus is correlated with morphological conservation or is linked to specific biological traits. Our analysis demonstrates that, in these organisms, it is possible to obtain high-quality draft genomes from single individuals with a relatively low sequencing effort. This result makes Lepidurus and Notostraca interesting models for genomic studies at taxonomic, ecological and evolutionary levels.

The availability of disease-specific genomic data is critical for developing new computational methods that predict the pathogenicity of human variants and advance the field of precision medicine. However, the lack of gold standards to properly train and benchmark such methods is one of the greatest challenges in the field. In response to this challenge, the scientific community is invited to participate in the Critical Assessment for Genome Interpretation (CAGI), where unpublished disease variants are available for classification by in silico methods. As part of the CAGI-5 challenge, we evaluated the performance of 18 submissions and three additional methods in predicting the pathogenicity of single nucleotide variants (SNVs) in checkpoint kinase 2 (CHEK2) for cases of breast cancer in Hispanic females. As part of the assessment, the efficacy of the analysis method and the setup of the challenge were also considered. The results indicated that though the challenge could benefit from additional participant data, the combined generalized linear model analysis and odds of pathogenicity analysis provided a framework to evaluate the methods submitted for SNV pathogenicity identification and for comparison to other available methods. The outcome of this challenge and the approaches used can help guide further advancements in identifying SNV-disease relationships.

The CAGI-5 pericentriolar material 1 (PCM1) challenge aimed to predict the effect of 38 transgenic human missense mutations in the PCM1 protein implicated in schizophrenia. Participants were provided with 16 benign variants (negative controls), 10 hypomorphic, and 12 loss of function variants. Six groups participated and were asked to predict the probability of effect and standard deviation associated to each mutation. Here, we present the challenge assessment. Prediction performance was evaluated using different measures to conclude in a final ranking which highlights the strengths and weaknesses of each group. The results show a great variety of predictions where some methods performed significantly better than others. Benign variants played an important role as negative controls, highlighting predictors biased to identify disease phenotypes. The best predictor, Bromberg lab, used a neural-network-based method able to discriminate between neutral and non-neutral single nucleotide polymorphisms. The CAGI-5 PCM1 challenge allowed us to evaluate the state of the art techniques for interpreting the effect of novel variants for a difficult target protein.

Thermodynamic stability is a fundamental property shared by all proteins. Changes in stability due to mutation are a widespread molecular mechanism in genetic diseases. Methods for the prediction of mutation-induced stability change have typically been developed and evaluated on incomplete and/or biased data sets. As part of the Critical Assessment of Genome Interpretation (CAGI), we explored the utility of high-throughput variant stability profiling (VSP) assay data as an alternative for the assessment of computational methods and evaluated state-of-the-art predictors against over 7,000 non-synonymous variants from two proteins. We found that predictions were modestly correlated with actual experimental values. Predictors fared better when evaluated as classifiers of extreme stability effects. While different methods emerged as top-performers depending on the metric, it is non-trivial to draw conclusions on their adoption or improvement. Our analyses revealed that only 16% of all variants in VSP assays could be confidently defined as stability-affecting. Furthermore, it is unclear to what extent VSP abundance scores were reasonable proxies for the stability-related quantities that participating methods were designed to predict. Overall, our observations underscore the need for clearly defined objectives when developing and using both computational and experimental methods in the context of measuring variant impact.

Accurate prediction of the impact of genomic variation on phenotype is a major goal of computational biology and an important contributor to personalized medicine. Computational predictions can lead to a better understanding of the mechanisms underlying genetic diseases, including cancer, but their adoption requires thorough and unbiased assessment. Cystathionine-beta-synthase (CBS) is an enzyme that catalyzes the first step of the transsulfuration pathway, from homocysteine to cystathionine, and in which variations are associated with human hyperhomocysteinemia and homocystinuria. We have created a computational challenge under the CAGI framework to evaluate how well different methods can predict the phenotypic effect(s) of CBS single amino acid substitutions using a blinded experimental data set. CAGI participants were asked to predict yeast growth based on the identity of the mutations. The performance of the methods was evaluated using several metrics. The CBS challenge highlighted the difficulty of predicting the phenotype of an ex vivo system in a model organism when classification models were trained on human disease data. We also discuss the variations in difficulty of prediction for known benign and deleterious variants, as well as identify methodological and experimental constraints with lessons to be learned for future challenges. ispartof: HUMAN MUTATION vol:40 issue:9 pages:1530-1545 ispartof: location:United States status: published

Additional file 1 Additional figures and tables referenced in the article.

Tosatto, Silvio/0000-0003-4525-7793; Zhang, Feng/0000-0003-3447-897X; Gonzalez, Jose Maria Fernandez/0000-0002-4806-5140; Devignes, Marie-Dominique/0000-0002-0399-8713; Wass, Mark/0000-0001-5428-6479; Falda, Marco/0000-0003-2642-519X; Thurlby, Natalie/0000-0002-1007-0286; Zosa, Elaine/0000-0003-2482-0663; Dessimoz, Christophe/0000-0002-2170-853X; Yunes, Jeffrey/0000-0003-1869-3231; Hamid, Md Nafiz/0000-0001-8681-6526; Hoehndorf, Robert/0000-0001-8149-5890; Dogan, Tunca/0000-0002-1298-9763; NOTARO, MARCO/0000-0003-4309-2200; Cozzetto, Domenico/0000-0001-6752-5432; Lewis, Kimberley/0000-0003-3010-8453; Roche, Daniel/0000-0002-9204-1840; Martin, Maria-Jesus/0000-0001-5454-2815; Tress, Michael/0000-0001-9046-6370; Tolvanen, Martti/0000-0003-3434-7646; Cheng, Jianlin/0000-0003-0305-2853; Rose, Peter/0000-0001-9981-9750; Renaux, Alexandre/0000-0002-4339-2791; Kacsoh, Balint/0000-0001-9171-0611; O'Donovan, Claire/0000-0001-8051-7429; Kulmanov, Maxat/0000-0003-1710-1820; Friedberg, Iddo/0000-0002-1789-8000; Zhou, Naihui/0000-0001-6268-6149, WOS: 000498615000001, PubMed ID: 31744546, Background The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. Results Here, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. Conclusion We conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens., National Science FoundationNational Science Foundation (NSF) [DBI1564756, DBI-1458359, DBI-1458390, DMS1614777, CMMI1825941, NSF 1458390]; Gordon and Betty Moore FoundationGordon and Betty Moore Foundation [GBMF 4552]; National Institutes of Health NIGMSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [P20 GM113132]; Cystic Fibrosis Foundation [CFRDP STANTO19R0]; BBSRCBiotechnology and Biological Sciences Research Council (BBSRC) [BB/K004131/1, BB/F00964X/1, BB/M025047/1, BB/M015009/1]; Consejo Nacional de Ciencia y Tecnologia Paraguay (CONACyT)Consejo Nacional de Ciencia y Tecnologia (CONACyT) [14-INV-088, PINV15-315]; NSFNational Science Foundation (NSF) [1660648, DBI 1759934, IIS1763246, DBI-1458477, 0965768, DMR-1420073, DBI-1458443]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01GM093123, DP1MH110234, UL1 TR002319, U24 TR002306]; Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy-EXC 2155 "RESIST"German Research Foundation (DFG) [39087428]; National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01GM123055, R01GM60595, R15GM120650, GM083107, GM116960, AI134678, NIH R35-GM128637, R00-GM097033]; ERCEuropean Research Council (ERC) [StG 757700]; Spanish Ministry of Science, Innovation and Universities [BFU2017-89833-P]; Severo Ochoa award; Centre of Excellence project "BioProspecting of Adriatic Sea"; Croatian Government; European Regional Development FundEuropean Union (EU) [KK.01.1.1.01.0002]; ATT Tieto kayttoon grant; Academy of FinlandAcademy of Finland; University of Turku; CSC-IT Center for Science Ltd.; University of Miami; National Cancer Institute of the National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [U01CA198942]; Helsinki Institute for Life Sciences; Academy of FinlandAcademy of Finland [292589]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China [31671367, 31471245, 91631301, 61872094, 61572139]; National Key Research and Development Program of China [2016YFC1000505, 2017YFC0908402]; Italian Ministry of Education, University and Research (MIUR) PRIN 2017 projectMinistry of Education, Universities and Research (MIUR) [2017483NH8]; Shanghai Municipal Science and Technology Major Project [2017SHZDZX01, 2018SHZDZX01]; UK Biotechnology and Biological Sciences Research CouncilBiotechnology and Biological Sciences Research Council (BBSRC) [BB/N019431/1, BB/L020505/1, BB/L002817/1]; Elsevier; Extreme Science and Engineering Discovery Environment (XSEDE) award [MCB160101, MCB160124]; Ministry of Education, Science and Technological Development of the Republic of Serbia [173001]; Taiwan Ministry of Science and Technology [106-2221-E-004-011-MY2]; Montana State University; Bavarian Ministry for Education; Simons Foundation; NIH NINDSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Neurological Disorders & Stroke (NINDS) [1R21NS103831-01]; University of Illinois at Chicago (UIC) Cancer Center award; UIC College of Liberal Arts and Sciences Faculty Award; UIC International Development Award; Yad Hanadiv [9660/2019]; National Institute of General Medical Science of the National Institute of Health [GM066099, GM079656]; Research Supporting Plan (PSR) of University of Milan [PSR2018-DIP-010-MFRAS]; Swiss National Science FoundationSwiss National Science Foundation (SNSF) [150654]; EMBL-European Bioinformatics Institute core funds; CAFA BBSRC [BB/N004876/1]; European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grantEuropean Union (EU) [778247]; COST ActionEuropean Cooperation in Science and Technology (COST) [BM1405]; NIH/NIGMSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [R01 GM071749]; National Human Genome Research Institute of the National of Health [U41 HG007234]; INB Grant (ISCIII-SGEFI/ERDF) [PT17/0009/0001]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [EEEAG-116E930]; KanSil [2016K121540]; Universita degli Studi di Milano; 111 ProjectMinistry of Education, China - 111 Project [B18015]; key project of Shanghai Science Technology [16JC1420402]; ZJLab; project Ribes Network POR-FESR 3S4H [TOPP-ALFREVE18-01]; PRID/SID of University of Padova [TOPP-SID19-01]; NIGMSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [R15GM120650]; King Abdullah University of Science and Technology (KAUST) Office of Sponsored Research (OSR) [URF/1/3454-01-01, URF/1/3790-01-01]; "the Human Project from Mind, Brain and Learning" of the NCCU Higher Education Sprout Project by the Taiwan Ministry of Education; National Center for High-performance ComputingIstanbul Technical University, The work of IF was funded, in part, by the National Science Foundation award DBI-1458359. The work of CSG and AJL was funded, in part, by the National Science Foundation award DBI-1458390 and GBMF 4552 from the Gordon and Betty Moore Foundation. The work of DAH and KAL was funded, in part, by the National Science Foundation award DBI-1458390, National Institutes of Health NIGMS P20 GM113132, and the Cystic Fibrosis Foundation CFRDP STANTO19R0. The work of AP, HY, AR, and MT was funded by BBSRC grants BB/K004131/1, BB/F00964X/1 and BB/M025047/1, Consejo Nacional de Ciencia y Tecnologia Paraguay (CONACyT) grants 14-INV-088 and PINV15-315, and NSF Advances in BioInformatics grant 1660648. The work of JC was partially supported by an NIH grant (R01GM093123) and two NSF grants (DBI 1759934 and IIS1763246). ACM acknowledges the support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy -EXC 2155 "RESIST" - Project ID 39087428. DK acknowledges the support from the National Institutes of Health (R01GM123055) and the National Science Foundation (DMS1614777, CMMI1825941). PB acknowledges the support from the National Institutes of Health (R01GM60595). GB and BZK acknowledge the support from the National Science Foundation (NSF 1458390) and NIH DP1MH110234. FS was funded by the ERC StG 757700 "HYPER-INSIGHT" and by the Spanish Ministry of Science, Innovation and Universities grant BFU2017-89833-P. FS further acknowledges the funding from the Severo Ochoa award to the IRB Barcelona. TS was funded by the Centre of Excellence project "BioProspecting of Adriatic Sea", co-financed by the Croatian Government and the European Regional Development Fund (KK.01.1.1.01.0002). The work of SK was funded by ATT Tieto kayttoon grant and Academy of Finland. JB and HM acknowledge the support of the University of Turku, the Academy of Finland and CSC -IT Center for Science Ltd. TB and SM were funded by the NIH awards UL1 TR002319 and U24 TR002306. The work of CZ and ZW was funded by the National Institutes of Health R15GM120650 to ZW and start-up funding from the University of Miami to ZW. The work of PWR was supported by the National Cancer Institute of the National Institutes of Health under Award Number U01CA198942. PR acknowledges NSF grant DBI-1458477. PT acknowledges the support from Helsinki Institute for Life Sciences. The work of AJM was funded by the Academy of Finland (No. 292589). The work of FZ and WT was funded by the National Natural Science Foundation of China (31671367, 31471245, 91631301) and the National Key Research and Development Program of China (2016YFC1000505, 2017YFC0908402]. CS acknowledges the support by the Italian Ministry of Education, University and Research (MIUR) PRIN 2017 project 2017483NH8. SZ is supported by the National Natural Science Foundation of China (No. 61872094 and No. 61572139) and Shanghai Municipal Science and Technology Major Project (No. 2017SHZDZX01). PLF and RLH were supported by the National Institutes of Health NIH R35-GM128637 and R00-GM097033. JG, DTJ, CW, DC, and RF were supported by the UK Biotechnology and Biological Sciences Research Council (BB/N019431/1, BB/L020505/1, and BB/L002817/1) and Elsevier. The work of YZ and CZ was funded in part by the National Institutes of Health award GM083107, GM116960, and AI134678; the National Science Foundation award DBI1564756; and the Extreme Science and Engineering Discovery Environment (XSEDE) award MCB160101 and MCB160124.; The work of BG, VP, RD, NS, and NV was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Project No. 173001. The work of YWL, WHL, and JMC was funded by the Taiwan Ministry of Science and Technology (106-2221-E-004-011-MY2). YWL, WHL, and JMC further acknowledge the support from "the Human Project from Mind, Brain and Learning" of the NCCU Higher Education Sprout Project by the Taiwan Ministry of Education and the National Center for High-performance Computing for computer time and facilities. The work of IK and AB was funded by Montana State University and NSF Advances in Biological Informatics program through grant number 0965768. BR, TG, and JR are supported by the Bavarian Ministry for Education through funding to the TUM. The work of RB, VG, MB, and DCEK was supported by the Simons Foundation, NIH NINDS grant number 1R21NS103831-01 and NSF award number DMR-1420073. CJJ acknowledges the funding from a University of Illinois at Chicago (UIC) Cancer Center award, a UIC College of Liberal Arts and Sciences Faculty Award, and a UIC International Development Award. The work of ML was funded by Yad Hanadiv (grant number 9660/2019). The work of OL and IN was funded by the National Institute of General Medical Science of the National Institute of Health through GM066099 and GM079656. Research Supporting Plan (PSR) of University of Milan number PSR2018-DIP-010-MFRAS. AWV acknowledges the funding from the BBSRC (CASE studentship BB/M015009/1). CD acknowledges the support from the Swiss National Science Foundation (150654). CO and MJM are supported by the EMBL-European Bioinformatics Institute core funds and the CAFA BBSRC BB/N004876/1. GG is supported by CAFA BBSRC BB/N004876/1. SCET acknowledges funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 778247 (IDPfun) and from COST Action BM1405 (NGP-net). SEB was supported by NIH/NIGMS grant R01 GM071749. The work of MLT, JMR, and JMF was supported by the National Human Genome Research Institute of the National of Health, grant numbers U41 HG007234. The work of JMF and JMR was also supported by INB Grant (PT17/0009/0001 - ISCIII-SGEFI/ERDF). VA acknowledges the funding from TUBITAK EEEAG-116E930. RCA acknowledges the funding from KanSil 2016K121540. GV acknowledges the funding from Universita degli Studi di Milano - Project "Discovering Patterns in Multi-Dimensional Data" and Project "Machine Learning and Big Data Analysis for Bioinformatics". SZ is supported by the National Natural Science Foundation of China (No. 61872094 and No. 61572139) and Shanghai Municipal Science and Technology Major Project (No. 2017SHZDZX01). RY and SY are supported by the 111 Project (NO. B18015), the key project of Shanghai Science & Technology (No. 16JC1420402), Shanghai Municipal Science and Technology Major Project (No. 2018SHZDZX01), and ZJLab. ST was supported by project Ribes Network POR-FESR 3S4H (No. TOPP-ALFREVE18-01) and PRID/SID of University of Padova (No. TOPP-SID19-01). CZ and ZW were supported by the NIGMS grant R15GM120650 to ZW and start-up funding from the University of Miami to ZW. The work of MK and RH was supported by the funding from King Abdullah University of Science and Technology (KAUST) Office of Sponsored Research (OSR) under Award No. URF/1/3454-01-01 and URF/1/3790-01-01. The work of SDM is funded, in part, by NSF award DBI-1458443.

In silico approaches are routinely adopted to predict the effects of genetic variants and their relation to diseases. The critical assessment of genome interpretation (CAGI) has established a common framework for the assessment of available predictors of variant effects on specific problems and our group has been an active participant of CAGI since its first edition. In this paper, we summarize our experience and lessons learned from the last edition of the experiment (CAGI-5). In particular, we analyze prediction performances of our tools on five CAGI-5 selected challenges grouped into three different categories: prediction of variant effects on protein stability, prediction of variant pathogenicity, and prediction of complex functional effects. For each challenge, we analyze in detail the performance of our tools, highlighting their potentialities and drawbacks. The aim is to better define the application boundaries of each tool.

Frataxin (FXN) is a highly conserved protein found in prokaryotes and eukaryotes that is required for efficient regulation of cellular iron homeostasis. Experimental evidence associates amino acid substitutions of the FXN to Friedreich Ataxia, a neurodegenerative disorder. Recently, new thermodynamic experiments have been performed to study the impact of somatic variations identified in cancer tissues on protein stability. The Critical Assessment of Genome Interpretation (CAGI) data provider at the University of Rome measured the unfolding free energy of a set of variants (FXN challenge data set) with far-UV circular dichroism and intrinsic fluorescence spectra. These values have been used to calculate the change in unfolding free energy between the variant and wild-type proteins at zero concentration of denaturant ( Δ Δ G H 2 O ) . The FXN challenge data set, composed of eight amino acid substitutions, was used to evaluate the performance of the current computational methods for predicting the Δ Δ G H 2 O value associated with the variants and to classify them as destabilizing and not destabilizing. For the fifth edition of CAGI, six independent research groups from Asia, Australia, Europe, and North America submitted 12 sets of predictions from different approaches. In this paper, we report the results of our assessment and discuss the limitations of the tested algorithms.

Here, we present BUSCA (http://busca.biocomp.unibo.it), a novel web server that integrates different computational tools for predicting protein subcellular localization. BUSCA combines methods for identifying signal and transit peptides (DeepSig and TPpred3), GPI-anchors (PredGPI) and transmembrane domains (ENSEMBLE3.0 and BetAware) with tools for discriminating subcellular localization of both globular and membrane proteins (BaCelLo, MemLoci and SChloro). Outcomes from the different tools are processed and integrated for annotating subcellular localization of both eukaryotic and bacterial protein sequences. We benchmark BUSCA against protein targets derived from recent CAFA experiments and other specific data sets, reporting performance at the state-of-the-art. BUSCA scores better than all other evaluated methods on 2732 targets from CAFA2, with a F1 value equal to 0.49 and among the best methods when predicting targets from CAFA3. We propose BUSCA as an integrated and accurate resource for the annotation of protein subcellular localization.

none 6 no Background: Modern genomic techniques allow to associate several Mendelian human diseases to single residue variations in different proteins. Molecular mechanisms explaining the relationship among genotype and phenotype are still under debate. Change of protein stability upon variation appears to assume a particular relevance in annotating whether a single residue substitution can or cannot be associated to a given disease. Thermodynamic properties of human proteins and of their disease related variants are lacking. In the present work, we take advantage of the available three dimensional structure of human proteins for predicting the role of disease related variations on the perturbation of protein stability. Results: We develop INPS3D, a new predictor based on protein structure for computing the effect of single residue variations on protein stability (ΔΔG), scoring at the state-of-the-art (Pearson's correlation value of the regression is equal to 0.72 with mean standard error of 1.15 kcal/mol on a blind test set comprising 351 variations in 60 proteins). We then filter 368 OMIM disease related proteins known with atomic resolution (where the three dimensional structure covers at least 70 % of the sequence) with 4717 disease related single residue variations and 685 polymorphisms without clinical consequence. We find that the effect on protein stability of disease related variations is larger than the effect of polymorphisms: in particular, by setting to |1 kcal/mol| the threshold between perturbing and not perturbing variations of the protein stability, about 44 % of disease related variations and 20 % of polymorphisms are predicted with |ΔΔG| > 1 kcal/mol, respectively. A consistent fraction of OMIM disease related variations is however predicted to promote |ΔΔG| ≤ 1 kcal/mol and we focus here on detecting features that can be associated to the thermodynamic property of the protein variant. Our analysis reveals that some 47 % of disease related variations promoting |ΔΔG| ≤ 1 are located in solvent exposed sites of the protein structure. We also find that the increase of the fraction of variations that in proteins are predicted with |ΔΔG| ≤ 1 kcal/mol, partially relates with the increasing number of the protein interacting partners, corroborating the notion that disease related, non-perturbing variations are likely to impair protein-protein interaction (70 % of the disease causing variations, with high accessible surface are indeed predicted in interacting sites). The set of OMIM surface accessible variations with |ΔΔG| ≤ 1 kcal/mol and located in interaction sites are 23 % of the total in 161 proteins. Among these, 43 proteins with some 327 disease causing variations are involved in signalling, structural biological processes, development and differentiation. Conclusions: We compute the effect of disease causing variations on protein stability with INPS3D, a new state-of-the-art tool for predicting the change in ΔΔG value associated to single residue substitution in protein structures. The analysis indicates that OMIM disease related variations in proteins promote a much larger effect on protein stability than polymorphisms non-associated to diseases. Disease related variations with a slight effect on protein stability (|ΔΔG| < 1 kcal/mol) frequently occur at the protein accessible surface suggesting that they are located in protein-protein interactions patches in putative human biological functional networks. The hypothesis is corroborated by proving that proteins with many disease related variations that slightly perturb protein stability are on average more connected in the human physical interactome (IntAct) than proteins with variations predicted with |ΔΔG| > 1 kcal/mol. Martelli, Pier Luigi; Fariselli, Piero; Savojardo, Castrense; Babbi, Giulia; Aggazio, Francesco; Casadio, Rita Martelli, Pier Luigi; Fariselli, Piero; Savojardo, Castrense; Babbi, Giulia; Aggazio, Francesco; Casadio, Rita

Background Recently, information derived by correlated mutations in proteins has regained relevance for predicting protein contacts. This is due to new forms of mutual information analysis that have been proven to be more suitable to highlight direct coupling between pairs of residues in protein structures and to the large number of protein chains that are currently available for statistical validation. It was previously discussed that disulfide bond topology in proteins is also constrained by correlated mutations. Results In this paper we exploit information derived from a corrected mutual information analysis and from the inverse of the covariance matrix to address the problem of the prediction of the topology of disulfide bonds in Eukaryotes. Recently, we have shown that Support Vector Regression (SVR) can improve the prediction for the disulfide connectivity patterns. Here we show that the inclusion of the correlated mutation information increases of 5 percentage points the SVR performance (from 54% to 59%). When this approach is used in combination with a method previously developed by us and scoring at the state of art in predicting both location and topology of disulfide bonds in Eukaryotes (DisLocate), the per-protein accuracy is 38%, 2 percentage points higher than that previously obtained. Conclusions In this paper we show that the inclusion of information derived from correlated mutations can improve the performance of the state of the art methods for predicting disulfide connectivity patterns in Eukaryotic proteins. Our analysis also provides support to the notion that improving methods to extract evolutionary information from multiple sequence alignments greatly contributes to the scoring performance of predictors suited to detect relevant features from protein chains.

MemPype is a Python-based pipeline including previously published methods for the prediction of signal peptides (SPEP), glycophosphatidylinositol (GPI) anchors (PredGPI), all-alpha membrane topology (ENSEMBLE), and a recent method (MemLoci) that specifically discriminates the localization of eukaryotic membrane proteins in: 'cell membrane', 'internal membranes', 'organelle membranes'. MemLoci scores with accuracy of 70% and generalized correlation coefficient (GCC) of 0.50 on a rigorous homology-unbiased validation set and overpasses other predictors for subcellular localization. The annotation process is based both on inheritance through homology and computational methods. Each submitted protein first retrieves, when available, up to 25 similar proteins (with sequence identity ≥50% and alignment coverage ≥50% on both sequences). This helps the identification of membrane-associated proteins and detailed localization tags. Each protein is also filtered for the presence of a GPI anchor [0.8% false positive rate (FPR)]. A positive score of GPI anchor prediction labels the sequence as exposed to 'Cell surface'. Concomitantly the sequence is analysed for the presence of a signal peptide and classified with MemLoci into one of three discriminated classes. Finally the sequence is filtered for predicting its putative all-alpha protein membrane topology (FPR