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Understanding the relationship between amino acid sequence and protein function is a long-standing challenge with far-reaching scientific and translational implications. State-of-the-art alignment-based techniques cannot predict function for one-third of microbial protein sequences, hampering our ability to exploit data from diverse organisms. Here, we train deep learning models to accurately predict functional annotations for unaligned amino acid sequences across rigorous benchmark assessments built from the 17,929 families of the protein families database Pfam. The models infer known patterns of evolutionary substitutions and learn representations that accurately cluster sequences from unseen families. Combining deep models with existing methods significantly improves remote homology detection, suggesting that the deep models learn complementary information. This approach extends the coverage of Pfam by9.5%, exceeding additions made over the last decade, and predicts function for 360 human reference proteome proteins with no previous Pfam annotation. These results suggest that deep learning models will be a core component of future protein annotation tools.

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions 15 megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.

Understanding the relationship between amino acid sequence and protein function is a long-standing problem in molecular biology with far-reaching scientific implications. Despite six decades of progress, state-of-the-art techniques cannot annotate 1/3 of microbial protein sequences, hampering our ability to exploit sequences collected from diverse organisms. In this paper, we explore an alternative methodology based on deep learning that learns the relationship between unaligned amino acid sequences and their functional annotations across all 17929 families of the Pfam database. Using the Pfam seed sequences we establish rigorous benchmark assessments that use both random and clustered data splits to control for potentially confounding sequence similarities between train and test sequences. Using Pfam full, we report convolutional networks that are significantly more accurate and computationally efficient than BLASTp, while learning sequence features such as structural disorder and transmembrane helices. Our model co-locates sequences from unseen families in embedding space, allowing sequences from novel families to be accurately annotated. These results suggest deep learning models will be a core component of future protein function prediction tools.

The major DNA sequencing technologies in use today produce either highly-accurate short reads or noisy long reads. We developed a protocol based on single-molecule, circular consensus sequencing (CCS) to generate highly-accurate (99.8%) long reads averaging 13.5 kb and applied it to sequence the well-characterized human HG002/NA24385. We optimized existing tools to comprehensively detect variants, achieving precision and recall above 99.91% for SNVs, 95.98% for indels, and 95.99% for structural variants. We estimate that 2,434 discordances are correctable mistakes in the high-quality Genome in a Bottle benchmark. Nearly all (99.64%) variants are phased into haplotypes, which further improves variant detection. De novo assembly produces a highly contiguous and accurate genome with contig N50 above 15 Mb and concordance of 99.998%. CCS reads match short reads for small variant detection, while enabling structural variant detection and de novo assembly at similar contiguity and markedly higher concordance than noisy long reads.

Copy number variants (CNVs) are an important type of genetic variation that play a causal role in many diseases. The ability to identify high quality CNVs is of substantial clinical relevance. However, CNVs are notoriously difficult to identify accurately from array-based methods and next-generation sequencing (NGS) data, particularly for small (< 10kbp) CNVs. Manual curation by experts widely remains the gold standard but cannot scale with the pace of sequencing, particularly in fast-growing clinical applications. We present the first proof-of-principle study demonstrating high throughput manual curation of putative CNVs by non-experts. We developed a crowdsourcing framework, called CrowdVariant, that leverages Google's high-throughput crowdsourcing platform to create a high confidence set of deletions for NA24385 (NIST HG002/RM 8391), an Ashkenazim reference sample developed in partnership with the Genome In A Bottle (GIAB) Consortium. We show that non-experts tend to agree both with each other and with experts on putative CNVs. We show that crowdsourced non-expert classifications can be used to accurately assign copy number status to putative CNV calls and identify 1,781 high confidence deletions in a reference sample. Multiple lines of evidence suggest these calls are a substantial improvement over existing CNV callsets and can also be useful in benchmarking and improving CNV calling algorithms. Our crowdsourcing methodology takes the first step toward showing the clinical potential for manual curation of CNVs at scale and can further guide other crowdsourcing genomics applications.

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of the heritability of this disease. Here, to test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole-genome sequencing in 2,657 European individuals with and without diabetes, and exome sequencing in 12,940 individuals from five ancestry groups. To increase statistical power, we expanded the sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support the idea that lower-frequency variants have a major role in predisposition to type 2 diabetes.

Here we present deep-learning techniques for healthcare, centering our discussion on deep learning in computer vision, natural language processing, reinforcement learning, and generalized methods. We describe how these computational techniques can impact a few key areas of medicine and explore how to build end-to-end systems. Our discussion of computer vision focuses largely on medical imaging, and we describe the application of natural language processing to domains such as electronic health record data. Similarly, reinforcement learning is discussed in the context of robotic-assisted surgery, and generalized deep-learning methods for genomics are reviewed.

MotivationInferring properties of biological sequences--such as determining the species-of-origin of a DNA sequence or the function of an amino-acid sequence--is a core task in many bioinformatics applications. These tasks are often solved using string-matching to map query sequences to labeled database sequences or via Hidden Markov Model-like pattern matching. In the current work we describe and assess an deep learning approach which trains a deep neural network (DNN) to predict database-derived labels directly from query sequences.ResultsWe demonstrate this DNN performs at state-of-the-art or above levels on a difficult, practically important problem: predicting species-of-origin from short reads of 16S ribosomal DNA. When trained on 16S sequences of over 13,000 distinct species, our DNN achieves read-level species classification accuracy within 2.0% of perfect memorization of training data, and produces more accurate genus-level assignments for reads from held-out species thank-mer, alignment, and taxonomic binning baselines. Moreover, our models exhibit greater robustness than these existing approaches to increasing noise in the query sequences. Finally, we show that these DNNs perform well on experimental 16S mock community dataset. Overall, our results constitute a first step towards our long-term goal of developing a general-purpose deep learning approach to predicting meaningful labels from short biological sequences.AvailabilityTensorFlow training code is available through GitHub (https://github.com/tensorflow/models/tree/master/research). Data in TensorFlow TFRecord format is available on Google Cloud Storage (gs://brain-genomics-public/research/seq2species/).Contactseq2species-interest@google.comSupplementary informationSupplementary data are available in a separate document.

The human genome is now investigated through high-throughput functional assays, and through the generation of population genomic data. These advances support the identification of functional genetic variants and the prediction of traits (e.g. deleterious variants and disease). This review summarizes lessons learned from the large-scale analyses of genome and exome data sets, modeling of population data and machine-learning strategies to solve complex genomic sequence regions. The review also portrays the rapid adoption of artificial intelligence/deep neural networks in genomics; in particular, deep learning approaches are well suited to model the complex dependencies in the regulatory landscape of the genome, and to provide predictors for genetic variant calling and interpretation.

Despite rapid advances in sequencing technologies, accurately calling genetic variants present in an individual genome from billions of short, errorful sequence reads remains challenging. Here we show that a deep convolutional neural network can call genetic variation in aligned next-generation sequencing read data by learning statistical relationships between images of read pileups around putative variant and true genotype calls. The approach, called DeepVariant, outperforms existing state-of-the-art tools. The learned model generalizes across genome builds and mammalian species, allowing nonhuman sequencing projects to benefit from the wealth of human ground-truth data. We further show that DeepVariant can learn to call variants in a variety of sequencing technologies and experimental designs, including deep whole genomes from 10X Genomics and Ion Ampliseq exomes, highlighting the benefits of using more automated and generalizable techniques for variant calling.

Comprehensive disease gene discovery in both common and rare diseases will require the efficient and accurate detection of all classes of genetic variation across tens to hundreds of thousands of human samples. We describe here a novel assembly-based approach to variant calling, the GATK HaplotypeCaller (HC) and Reference Confidence Model (RCM), that determines genotype likelihoods independently per-sample but performs joint calling across all samples within a project simultaneously. We show by calling over 90,000 samples from the Exome Aggregation Consortium (ExAC) that, in contrast to other algorithms, the HC-RCM scales efficiently to very large sample sizes without loss in accuracy; and that the accuracy of indel variant calling is superior in comparison to other algorithms. More importantly, the HC-RCM produces a fully squared-off matrix of genotypes across all samples at every genomic position being investigated. The HC-RCM is a novel, scalable, assembly-based algorithm with abundant applications for population genetics and clinical studies.

Germline mutation detection from human DNA sequence data is challenging due to the rarity of such events relative to the intrinsic error rates of sequencing technologies and the uneven coverage across the genome. We developed PhaseByTransmission (PBT) to identify de novo single nucleotide variants and short insertions and deletions (indels) from sequence data collected in parent-offspring trios. We compute the joint probability of the data given the genotype likelihoods in the individual family members, the known familial relationships and a prior probability for the mutation rate. Candidate de novo mutations (DNMs) are reported along with their posterior probability, providing a systematic way to prioritize them for validation. Our tool is integrated in the Genome Analysis Toolkit and can be used together with the ReadBackedPhasing module to infer the parental origin of DNMs based on phase-informative reads. Using simulated data, we show that PBT outperforms existing tools, especially in low coverage data and on the X chromosome. We further show that PBT displays high validation rates on empirical parent-offspring sequencing data for whole-exome data from 104 trios and X-chromosome data from 249 parent-offspring families. Finally, we demonstrate an association between father’s age at conception and the number of DNMs in female offspring’s X chromosome, consistent with previous literature reports.European Journal of Human Genetics advance online publication, 23 November 2016; doi:10.1038/ejhg.2016.147.

Spontaneously arising (de novo) mutations have an important role in medical genetics. For diseases with extensive locus heterogeneity, such as autism spectrum disorders (ASDs), the signal from de novo mutations is distributed across many genes, making it difficult to distinguish disease-relevant mutations from background variation. Here we provide a statistical framework for the analysis of excesses in de novo mutation per gene and gene set by calibrating a model of de novo mutation. We applied this framework to de novo mutations collected from 1,078 ASD family trios, and, whereas we affirmed a significant role for loss-of-function mutations, we found no excess of de novo loss-of-function mutations in cases with IQ above 100, suggesting that the role of de novo mutations in ASDs might reside in fundamental neurodevelopmental processes. We also used our model to identify ∼1,000 genes that are significantly lacking in functional coding variation in non-ASD samples and are enriched for de novo loss-of-function mutations identified in ASD cases.

Next-generation sequencing (NGS) is a rapidly evolving set of technologies that can be used to determine the sequence of an individual’s genome1 by calling genetic variants present in an individual using billions of short, errorful sequence reads2. Despite more than a decade of effort and thousands of dedicated researchers, the hand-crafted and parameterized statistical models used for variant calling still produce thousands of errors and missed variants in each genome3,4. Here we show that a deep convolutional neural network5 can call genetic variation in aligned next-generation sequencing read data by learning statistical relationships (likelihoods) between images of read pileups around putative variant sites and ground-truth genotype calls. This approach, called DeepVariant, outperforms existing tools, even winning the “highest performance” award for SNPs in a FDA-administered variant calling challenge. The learned model generalizes across genome builds and even to other mammalian species, allowing non-human sequencing projects to benefit from the wealth of human ground truth data. We further show that, unlike existing tools which perform well on only a specific technology, DeepVariant can learn to call variants in a variety of sequencing technologies and experimental designs, from deep whole genomes from 10X Genomics to Ion Ampliseq exomes. DeepVariant represents a significant step from expert-driven statistical modeling towards more automatic deep learning approaches for developing software to interpret biological instrumentation data.

SummaryLarge-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) sequence data for 60,706 individuals of diverse ethnicities generated as part of the Exome Aggregation Consortium (ExAC). The resulting catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We show that this catalogue can be used to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; we identify 3,230 genes with near-complete depletion of truncating variants, 72% of which have no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human “knockout” variants in protein-coding genes.

SummaryTo characterize the role of rare complete human knockouts in autism spectrum disorders (ASDs), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a 2-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudoautosomal regions on the X chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together, these results provide compelling evidence that rare autosomal and X chromosome complete gene knockouts are important inherited risk factors for ASD.

International audience; Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.

Defective Gene Detective Identifying genes that give rise to diseases is one of the major goals of sequencing human genomes. However, putative loss-of-function genes, which are often some of the first identified targets of genome and exome sequencing, have often turned out to be sequencing errors rather than true genetic variants. In order to identify the true scope of loss-of-function genes within the human genome, MacArthur et al. (p. 823 ; see the Perspective by Quintana-Murci ) extensively validated the genomes from the 1000 Genomes Project, as well as an additional European individual, and found that the average person has about 100 true loss-of-function alleles of which approximately 20 have two copies within an individual. Because many known disease-causing genes were identified in “normal” individuals, the process of clinical sequencing needs to reassess how to identify likely causative alleles.

Recent advances in sequencing technology make it possible to comprehensively catalog genetic variation in population samples, creating a foundation for understanding human disease, ancestry and evolution. The amounts of raw data produced are prodigious, and many computational steps are required to translate this output into high-quality variant calls. We present a unified analytic framework to discover and genotype variation among multiple samples simultaneously that achieves sensitive and specific results across five sequencing technologies and three distinct, canonical experimental designs. Our process includes (i) initial read mapping; (ii) local realignment around indels; (iii) base quality score recalibration; (iv) SNP discovery and genotyping to find all potential variants; and (v) machine learning to separate true segregating variation from machine artifacts common to next-generation sequencing technologies. We here discuss the application of these tools, instantiated in the Genome Analysis Toolkit, to deep whole-genome, whole-exome capture and multi-sample low-pass (∼4×) 1000 Genomes Project datasets.

Exome sequencing is a powerful tool for discovery of the Mendelian disease genes. Previously, we reported a novel locus for autosomal recessive non-syndromic mental retardation (NSMR) in a consanguineous family [Nolan, D.K., Chen, P., Das, S., Ober, C. and Waggoner, D. (2008) Fine mapping of a locus for nonsyndromic mental retardation on chromosome 19p13. Am. J. Med. Genet. A, 146A, 1414–1422]. Using linkage and homozygosity mapping, we previously localized the gene to chromosome 19p13. The parents of this sibship were recently included in an exome sequencing project. Using a series of filters, we narrowed the putative causal mutation to a single variant site that segregated with NSMR: the mutation was homozygous in five affected siblings but in none of eight unaffected siblings. This mutation causes a substitution of a leucine for a highly conserved proline at amino acid 182 in TECR (trans-2,3-enoyl-CoA reductase), a synaptic glycoprotein. Our results reveal the value of massively parallel sequencing for identification of novel disease genes that could not be found using traditional approaches and identifies only the seventh causal mutation for autosomal recessive NSMR.

We sequenced all protein-coding regions of the genome (the "exome") in two family members with combined hypolipidemia, marked by extremely low plasma levels of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides. These two participants were compound heterozygotes for two distinct nonsense mutations in ANGPTL3 (encoding the angiopoietin-like 3 protein). ANGPTL3 has been reported to inhibit lipoprotein lipase and endothelial lipase, thereby increasing plasma triglyceride and HDL cholesterol levels in rodents. Our finding of ANGPTL3 mutations highlights a role for the gene in LDL cholesterol metabolism in humans and shows the usefulness of exome sequencing for identification of novel genetic causes of inherited disorders. (Funded by the National Human Genome Research Institute and others.).

Over the past decade, attempts to explain the unusual size and prevalence of low-complexity regions (LCRs) in the proteins of the human malaria parasite Plasmodium falciparum have used both neutral and adaptive models. This past research has offered conflicting explanations for LCR characteristics and their role in, and influence on, the evolution of genome structure. Here we show that P. falciparum LCRs (PfLCRs) are not a single phenomenon, but rather consist of at least three distinct types of sequence, and this heterogeneity is the source of the conflict in the literature. Using molecular and population genetics, we show that these families of PfLCRs are evolving by different mechanisms. One of these families, named here the HighGC family, is of particular interest because these LCRs act as recombination hotspots, both in genes under positive selection for high levels of diversity which can be created by recombination (antigens) and those likely to be evolving neutrally or under negative selection (metabolic enzymes). We discuss how the discovery of these distinct species of PfLCRs helps to resolve previous contradictory studies on LCRs in malaria and contributes to our understanding of the evolution of the of the parasite's unusual genome.

In this commentary we discuss recent progress in our understanding of adaptive protein evolution. We begin with a general introduction to proteins and their evolution, quickly focusing on the question of how natural selection produces proteins with novel functions. We then summarize the theory of latent protein adaptation advanced by the joint articles by Amitai et al. (2007), HFSP J. 1, 67–78 and Wroe et al. (2007) HFSP J. 1, 79–87, published in the first issue edition of the HFSP Journal. This theory provides a biophysical framework linking the effects of individual mutations on promiscuous protein function, neutral genetic drift, and gene duplication to the process of adaptive protein evolution.

The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies., Wellcome Trust (London, England) (Core Award 090532/Z/09/Z), Wellcome Trust (London, England) (Senior Investigator Award 095552/Z/11/Z ), Wellcome Trust (London, England) (WT095908), Wellcome Trust (London, England) (WT109497), Wellcome Trust (London, England) (WT098051), Wellcome Trust (London, England) (WT086084/Z/08/Z), Wellcome Trust (London, England) (WT100956/Z/13/Z ), Wellcome Trust (London, England) (WT097307), Wellcome Trust (London, England) (WT0855322/Z/08/Z ), Wellcome Trust (London, England) (WT090770/Z/09/Z ), Wellcome Trust (London, England) (Major Overseas program in Vietnam grant 089276/Z.09/Z), Medical Research Council (Great Britain) (grant G0801823), Biotechnology and Biological Sciences Research Council (Great Britain) (grant BB/I02593X/1), Biotechnology and Biological Sciences Research Council (Great Britain) (grant BB/I021213/1), Zhongguo ke xue ji shu qing bao yan jiu suo. Office of 863 Programme of China (2012AA02A201), National Basic Research Program of China (2011CB809201), National Basic Research Program of China (2011CB809202), National Basic Research Program of China (2011CB809203), National Natural Science Foundation of China (31161130357), Shenzhen Municipal Government of China (grant ZYC201105170397A), Canadian Institutes of Health Research (grant 136855), Quebec Ministry of Economic Development, Innovation, and Exports (PSR-SIIRI-195), Germany. Bundesministerium für Bildung und Forschung (0315428A), Germany. Bundesministerium für Bildung und Forschung (01GS08201), Germany. Bundesministerium für Bildung und Forschung (BMBF-EPITREAT grant 0316190A), Deutsche Forschungsgemeinschaft (Emmy Noether Grant KO4037/1-1), Beatriu de Pinos Program (2006 BP-A 10144), Beatriu de Pinos Program (2009 BP-B 00274), Spanish National Institute for Health (grant PRB2 IPT13/0001-ISCIII-SGEFI/FEDER), Japan Society for the Promotion of Science (fellowship number PE13075), Marie Curie Actions Career Integration (grant 303772), Fonds National Suisse del la Recherche, SNSF, Scientifique (31003A_130342), National Center for Biotechnology Information (U.S.) (U54HG3067), National Center for Biotechnology Information (U.S.) (U54HG3273), National Center for Biotechnology Information (U.S.) (U01HG5211), National Center for Biotechnology Information (U.S.) (U54HG3079), National Center for Biotechnology Information (U.S.) (R01HG2898), National Center for Biotechnology Information (U.S.) (R01HG2385), National Center for Biotechnology Information (U.S.) (RC2HG5552), National Center for Biotechnology Information (U.S.) (U01HG6513), National Center for Biotechnology Information (U.S.) (U01HG5214), National Center for Biotechnology Information (U.S.) (U01HG5715), National Center for Biotechnology Information (U.S.) (U01HG5718), National Center for Biotechnology Information (U.S.) (U01HG5728), National Center for Biotechnology Information (U.S.) (U41HG7635), National Center for Biotechnology Information (U.S.) (U41HG7497), National Center for Biotechnology Information (U.S.) (R01HG4960), National Center for Biotechnology Information (U.S.) (R01HG5701), National Center for Biotechnology Information (U.S.) (R01HG5214), National Center for Biotechnology Information (U.S.) (R01HG6855), National Center for Biotechnology Information (U.S.) (R01HG7068), National Center for Biotechnology Information (U.S.) (R01HG7644), National Center for Biotechnology Information (U.S.) (DP2OD6514), National Center for Biotechnology Information (U.S.) (DP5OD9154), National Center for Biotechnology Information (U.S.) (R01CA166661), National Center for Biotechnology Information (U.S.) (R01CA172652), National Center for Biotechnology Information (U.S.) (P01GM99568), National Center for Biotechnology Information (U.S.) (R01GM59290), National Center for Biotechnology Information (U.S.) (R01GM104390), National Center for Biotechnology Information (U.S.) (T32GM7790), National Center for Biotechnology Information (U.S.) (R01HL87699), National Center for Biotechnology Information (U.S.) (R01HL104608), National Center for Biotechnology Information (U.S.) (T32HL94284), National Center for Biotechnology Information (U.S.) (HHSN268201100040C), National Center for Biotechnology Information (U.S.) (HHSN272201000025C), Lundbeck Foundation (grant R170-2014-1039, Simons Foundation (SFARI award SF51), National Science Foundation (U.S.) (Research Fellowship DGE-1147470)

Protein sequences frequently contain regions composed of a reduced number of amino acids. Despite their presence in about half of all proteins and their unusual prevalence in the malaria parasite Plasmodium falciparum, the function and evolution of such low-complexity regions (LCRs) remain unclear. Here we show that LCR abundance and amino acid composition depend largely, but not exclusively, on genomic A+T content and obey power–law growth dynamics. Further, our results indicate that LCRs are analogous to microsatellites in that DNA replication slippage and unequal crossover recombination are important molecular mechanisms for LCR expansion. We support this hypothesis by demonstrating that the size of LCR insertions/deletions among orthologous genes depends upon length. Moreover, we show that LCRs enable intra-exonic recombination in a key family of cell-surface antigens in P. falciparum and thus likely facilitate the generation of antigenic diversity. We conclude with a mechanistic model for LCR evolution that links the pattern of LCRs within P. falciparum to its high genomic A+T content and recombination rate.

Different experimental structures of the same protein or of proteins with high sequence similarity contain many small variations. Here we construct ensembles of “high-sequence similarity Protein Data Bank” (HSP) structures and consider the extent to which such ensembles represent the structural heterogeneity of the native state in solution. We find that different NMR measurements probing structure and dynamics of given proteins in solution, including order parameters, scalar couplings, and residual dipolar couplings, are remarkably well reproduced by their respective high-sequence similarity Protein Data Bank ensembles; moreover, we show that the effects of uncertainties in structure determination are insufficient to explain the results. These results highlight the importance of accounting for native-state protein dynamics in making comparisons with ensemble-averaged experimental data and suggest that even a modest number of structures of a protein determined under different conditions, or with small variations in sequence, capture a representative subset of the true native-state ensemble.

We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy--for obtaining experimental information at the atomic level about the structural and dynamical features of proteins--with the ability of molecular dynamics simulations to explore a wide range of protein conformations. We illustrate the method for human ubiquitin in solution and find that there is considerable conformational heterogeneity throughout the protein structure. The interior atoms of the protein are tightly packed in each individual conformation that contributes to the ensemble but their overall behaviour can be described as having a significant degree of liquid-like character. The protocol is completely general and should lead to significant advances in our ability to understand and utilize the structures of native proteins.

Proteins are dynamic molecules, exhibiting structural heterogeneity in the form of anisotropic motion and discrete conformational substates, often of functional importance. In protein structure determination by X-ray crystallography, the observed diffraction pattern results from the scattering of X-rays by an ensemble of heterogeneous molecules, ordered and oriented by packing in a crystal lattice. The majority of proteins diffract to resolutions where heterogeneity is difficult to identify and model, and are therefore approximated by a single, average conformation with isotropic variance. Here we show that disregarding structural heterogeneity introduces degeneracy into the structure determination process, as many single, isotropic models exist that explain the diffraction data equally well. The large differences among these models imply that the accuracy of crystallographic structures has been widely overestimated. Further, it suggests that analyses that depend on small differences in the relative positions of atoms may be flawed.

We compare the modelling accuracy of two common rotamer libraries, the Dunbrack-Cohen and the 'Penultimate' rotamer libraries, with that of a novel library of discrete side chain conformations extracted from the Protein Data Bank. These side chain conformer libraries are extracted automatically from high-quality protein structures using stringent filters and maintain crystallographic bond lengths and angles. This contrasts with traditional rotamer libraries defined in terms of chi angles under the assumption of idealized covalent geometry. We demonstrate that side chain modelling onto native and near-native main chain conformations is significantly more successful with the conformer libraries than with the rotamer libraries when solely considering excluded-volume interactions. The rotamer libraries are inadequate to model side chains without atomic clashes on over 20% of targets if the backbone is held fixed in the native conformation. An algorithm is described for simultaneously modelling both main chain and side chain atoms during discrete ab initio sampling. The resulting models have equivalent root mean square deviations from the experimentally determined protein loops as models from backbone-only ensembles, indicating that all-atom modelling does not detract from the accuracy of conformational sampling.

We present a novel de novo method to generate protein models from sparse, discretized restraints on the conformation of the main chain and side chain atoms. We focus on Calpha-trace generation, the problem of constructing an accurate and complete model from approximate knowledge of the positions of the Calpha atoms and, in some cases, the side chain centroids. Spatial restraints on the Calpha atoms and side chain centroids are supplemented by constraints on main chain geometry, phi/xi angles, rotameric side chain conformations, and inter-atomic separations derived from analyses of known protein structures. A novel conformational search algorithm, combining features of tree-search and genetic algorithms, generates models consistent with these restraints by propensity-weighted dihedral angle sampling. Models with ideal geometry, good phi/xi angles, and no inter-atomic overlaps are produced with 0.8 A main chain and, with side chain centroid restraints, 1.0 A all-atom root-mean-square deviation (RMSD) from the crystal structure over a diverse set of target proteins. The mean model derived from 50 independently generated models is closer to the crystal structure than any individual model, with 0.5 A main chain RMSD under only Calpha restraints and 0.7 A all-atom RMSD under both Calpha and centroid restraints. The method is insensitive to randomly distributed errors of up to 4 A in the Calpha restraints. The conformational search algorithm is efficient, with computational cost increasing linearly with protein size. Issues relating to decoy set generation, experimental structure determination, efficiency of conformational sampling, and homology modeling are discussed.

We describe a novel method to generate ensembles of conformations of the main-chain atoms [N, C(alpha), C, O, Cbeta] for a sequence of amino acids within the context of a fixed protein framework. Each conformation satisfies fundamental stereo-chemical restraints such as idealized geometry, favorable phi/psi angles, and excluded volume. The ensembles include conformations both near and far from the native structure. Algorithms for effective conformational sampling and constant time overlap detection permit the generation of thousands of distinct conformations in minutes. Unlike previous approaches, our method samples dihedral angles from fine-grained phi/psi state sets, which we demonstrate is superior to exhaustive enumeration from coarse phi/psi sets. Applied to a large set of loop structures, our method samples consistently near-native conformations, averaging 0.4, 1.1, and 2.2 A main-chain root-mean-square deviations for four, eight, and twelve residue long loops, respectively. The ensembles make ideal decoy sets to assess the discriminatory power of a selection method. Using these decoy sets, we conclude that quality of anchor geometry cannot reliably identify near-native conformations, though the selection results are comparable to previous loop prediction methods. In a subsequent study (de Bakker et al.: Proteins 2003;51:21-40), we demonstrate that the AMBER forcefield with the Generalized Born solvation model identifies near-native conformations significantly better than previous methods.

Elevated low-density lipoprotein cholesterol (LDL-C) is a treatable, heritable risk factor for cardiovascular disease. Genome-wide association studies (GWASs) have identified 157 variants associated with lipid levels but are not well suited to assess the impact of rare and low-frequency variants. To determine whether rare or low-frequency coding variants are associated with LDL-C, we exome sequenced 2,005 individuals, including 554 individuals selected for extreme LDL-C (>98(th) or

Schizophrenia is a common disease with a complex aetiology, probably involving multiple and heterogeneous genetic factors. Here, by analysing the exome sequences of 2,536 schizophrenia cases and 2,543 controls, we demonstrate a polygenic burden primarily arising from rare (less than 1 in 10,000), disruptive mutations distributed across many genes. Particularly enriched gene sets include the voltage-gated calcium ion channel and the signalling complex formed by the activity-regulated cytoskeleton-associated scaffold protein (ARC) of the postsynaptic density, sets previously implicated by genome-wide association and copy-number variation studies. Similar to reports in autism, targets of the fragile X mental retardation protein (FMRP, product of FMR1) are enriched for case mutations. No individual gene-based test achieves significance after correction for multiple testing and we do not detect any alleles of moderately low frequency (approximately 0.5 to 1 per cent) and moderately large effect. Taken together, these data suggest that population-based exome sequencing can discover risk alleles and complements established gene-mapping paradigms in neuropsychiatric disease., National Human Genome Research Institute (U.S.) (Grant U54HG003067)

Background Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. Methods We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. Results An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G -> A and IVS3+1G -> T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P

Finnish samples have been extensively utilized in studying single-gene disorders, where the founder effect has clearly aided in discovery, and more recently in genome-wide association studies of complex traits, where the founder effect has had less obvious impacts. As the field starts to explore rare variants' contribution to polygenic traits, it is of great importance to characterize and confirm the Finnish founder effect in sequencing data and to assess its implications for rare-variant association studies. Here, we employ forward simulation, guided by empirical deep resequencing data, to model the genetic architecture of quantitative polygenic traits in both the general European and the Finnish populations simultaneously. We demonstrate that power of rare-variant association tests is higher in the Finnish population, especially when variants' phenotypic effects are tightly coupled with fitness effects and therefore reflect a greater contribution of rarer variants. SKAT-O, variable-threshold tests, and single-variant tests are more powerful than other rare-variant methods in the Finnish population across a range of genetic models. We also compare the relative power and efficiency of exome array genotyping to those of high-coverage exome sequencing. At a fixed cost, less expensive genotyping strategies have far greater power than sequencing; in a fixed number of samples, however, genotyping arrays miss a substantial portion of genetic signals detected in sequencing, even in the Finnish founder population. As genetic studies probe sequence variation at greater depth in more diverse populations, our simulation approach provides a framework for evaluating various study designs for gene discovery.

Adaptation is often regarded as the sequential fixation of individually, intrinsically beneficial mutations. Contrary to this expectation, we find a surprisingly large number of evolutionary trajectories on which natural selection first favors a mutation, then favors its removal, and later still favors its ultimate restoration during the course of antibiotic resistance evolution. The existence of reversion trajectories implies that natural selection may not follow the most parsimonious path separating two alleles, even during adaptation. Altogether, this discovery highlights the unusual and potentially circuitous routes natural selection can follow during adaptation.

This unit describes how to use BWA and the Genome Analysis Toolkit (GATK) to map genome sequencing data to a reference and produce high-quality variant calls that can be used in downstream analyses. The complete workflow includes the core NGS data processing steps that are necessary to make the raw data suitable for analysis by the GATK, as well as the key methods involved in variant discovery using the GATK.

We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD., Author Summary This study evaluates association of rare variants and autism spectrum disorders (ASD) in case and control samples sequenced by two centers. Before doing association analyses, we studied how to combine information across studies. We first harmonized the whole-exome sequence (WES) data, across centers, in terms of the distribution of rare variation. Key features included filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. After filtering, the vast majority of variants calls from seven samples sequenced at both centers matched. We also evaluated whether one should combine summary statistics from data from each center (meta-analysis) or combine data and analyze it together (mega-analysis). For many gene-based tests, we showed that mega-analysis yields more power. After quality control of data from 1,039 ASD cases and 870 controls and a range of analyses, no gene showed exome-wide evidence of significant association. Our results comport with recent results demonstrating that hundreds of genes affect risk for ASD; they suggest that rare risk variants are scattered across these many genes, and thus larger samples will be required to identify those genes.

Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

Kevin Davies, the (co-)author of Cracking the Genome and Breakthrough, brings his popularization of modern genetics to the newly emerging field of personal genomics and next-generation DNA sequencing (NGS) in his most recent book, The $1000 Genome: The Revolution in DNA Sequencing and the New Era of Personalized Medicine. Davies's newest work recounts the post-Human Genome Project (HGP) quest for the ever less expensive human genome sequence and its reciprocal pursuit of the meaning of variation among human genomes for traits, particularly common disease. Weaving these two disparate threads together throughout the book, Davies provides a chronologically anchored story of the development of next-generation DNA sequencing technology, from Life/454 and Illumina to Pacific Biosciences, personalized genomics from Watson and Venter to the Public Genome Project, and whole-genome association studies linking SNPs to common human diseases.Although the broad range of topics ensures that each chapter introduces an interesting big idea, technology, or social issue, the narrative flow can be jarring as Davies jumps around among NGS technologies, personal genomics companies, and medical genetics research. The book relies perhaps too heavily on colorful stories of the companies, projects, and people pursuing the $1,000 genome, especially given the limited discussions and analyses of the science and technology itself. Nevertheless, The $1,000 Genome does a good job presenting the complex and interconnected technologies and science underlying the genomics revolution as vignettes accessible to an informed but nonexpert audience. After a childhood of reading popular science books about physics, The $1,000 Genome delivers exactly what I have come to expect of such books: an enjoyable, easy read that makes one wish they were part of the exciting story itself, which for me is a fact that I'm thankful for even more after reading Davies's book.

Background Comprehensive sequence characterization across the MHC is important for successful organ transplantation and genetic association studies. To this end, we have developed an automated sample preparation, molecular barcoding and multiplexing protocol for the amplification and sequence-determination of class I HLA loci. We have coupled this process to a novel HLA calling algorithm to determine the most likely pair of alleles at each locus. Results We have benchmarked our protocol with 270 HapMap individuals from four worldwide populations with 96.4% accuracy at 4-digit resolution. A variation of this initial protocol, more suitable for large sample sizes, in which molecular barcodes are added during PCR rather than library construction, was tested on 95 HapMap individuals with 98.6% accuracy at 4-digit resolution. Conclusions Next-generation sequencing on the 454 FLX Titanium platform is a reliable, efficient, and scalable technology for HLA typing.

Next-generation DNA sequencing (NGS) projects, such as the 1000 Genomes Project, are already revolutionizing our understanding of genetic variation among individuals. However, the massive data sets generated by NGS—the 1000 Genome pilot alone includes nearly five terabases—make writing feature-rich, efficient, and robust analysis tools difficult for even computationally sophisticated individuals. Indeed, many professionals are limited in the scope and the ease with which they can answer scientific questions by the complexity of accessing and manipulating the data produced by these machines. Here, we discuss our Genome Analysis Toolkit (GATK), a structured programming framework designed to ease the development of efficient and robust analysis tools for next-generation DNA sequencers using the functional programming philosophy of MapReduce. The GATK provides a small but rich set of data access patterns that encompass the majority of analysis tool needs. Separating specific analysis calculations from common data management infrastructure enables us to optimize the GATK framework for correctness, stability, and CPU and memory efficiency and to enable distributed and shared memory parallelization. We highlight the capabilities of the GATK by describing the implementation and application of robust, scale-tolerant tools like coverage calculators and single nucleotide polymorphism (SNP) calling. We conclude that the GATK programming framework enables developers and analysts to quickly and easily write efficient and robust NGS tools, many of which have already been incorporated into large-scale sequencing projects like the 1000 Genomes Project and The Cancer Genome Atlas., National Human Genome Research Institute (U.S.) (Large Scale Sequencing and Analysis of Genomes grant (54 HG003067)), National Human Genome Research Institute (U.S.) (Joint SNP and CNV calling in 1000 Genomes sequence data grant (U01 HG005208))

Antibiotic resistance arises through mechanisms such as selection of naturally occurring resistant mutants and horizontal gene transfer. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here we show that sub-lethal levels of bactericidal antibiotics induce mutagenesis, resulting in heterogeneous increases in the minimum inhibitory concentration for a range of antibiotics, irrespective of the drug target. This increase in mutagenesis correlates with an increase in ROS, and is prevented by the ROS scavenger thiourea and by anaerobic conditions, indicating that sub-lethal concentrations of antibiotics induce mutagenesis by stimulating the production of ROS. We demonstrate that these effects can lead to mutant strains that are sensitive to the applied antibiotic but resistant to other antibiotics. This work establishes a radical-based molecular mechanism whereby sub-lethal levels of antibiotics can lead to multidrug resistance, which has important implications for the widespread use and misuse of antibiotics.