A genomic mutational constraint map using variation in 76,156 human genomes.

The depletion of disruptive variation caused by purifying natural selection (constraint) has been widely used to investigate protein-coding genes underlying human disorders1-4, but attempts to assess constraint for non-protein-coding regions have proved more difficult. Here we aggregate, process and release a dataset of 76,156 human genomes from the Genome Aggregation Database (gnomAD)-the largest public open-access human genome allele frequency reference dataset-and use it to build a genomic constraint map for the whole genome (genomic non-coding constraint of haploinsufficient variation (Gnocchi)). We present a refined mutational model that incorporates local sequence context and regional genomic features to detect depletions of variation. As expected, the average constraint for protein-coding sequences is stronger than that for non-coding regions. Within the non-coding genome, constrained regions are enriched for known regulatory elements and variants that are implicated in complex human diseases and traits, facilitating the triangulation of biological annotation, disease association and natural selection to non-coding DNA analysis. More constrained regulatory elements tend to regulate more constrained protein-coding genes, which in turn suggests that non-coding constraint can aid the identification of constrained genes that are as yet unrecognized by current gene constraint metrics. We demonstrate that this genome-wide constraint map improves the identification and interpretation of functional human genetic variation.

A structural variation reference for medical and population genetics.

Structural variants (SVs) rearrange large segments of DNA1 and can have profound consequences in evolution and human disease2,3. As national biobanks, disease-association studies, and clinical genetic testing have grown increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD)4 have become integral in the interpretation of single-nucleotide variants (SNVs)5. However, there are no reference maps of SVs from high-coverage genome sequencing comparable to those for SNVs. Here we present a reference of sequence-resolved SVs constructed from 14,891 genomes across diverse global populations (54% non-European) in gnomAD. We discovered a rich and complex landscape of 433,371 SVs, from which we estimate that SVs are responsible for 25-29% of all rare protein-truncating events per genome. We found strong correlations between natural selection against damaging SNVs and rare SVs that disrupt or duplicate protein-coding sequence, which suggests that genes that are highly intolerant to loss-of-function are also sensitive to increased dosage6. We also uncovered modest selection against noncoding SVs in cis-regulatory elements, although selection against protein-truncating SVs was stronger than all noncoding effects. Finally, we identified very large (over one megabase), rare SVs in 3.9% of samples, and estimate that 0.13% of individuals may carry an SV that meets the existing criteria for clinically important incidental findings7. This SV resource is freely distributed via the gnomAD browser8 and will have broad utility in population genetics, disease-association studies, and diagnostic screening.

Nondestructive adult age at death estimation: Visualizing cementum annulations in a known age historical human assemblage using synchrotron X-ray microtomography.

OBJECTIVES: Adult age at death estimation continues to challenge physical anthropologists. One estimation method involves counting tooth cementum annulations (TCA). Non-destructively accessing TCA is a critical step to approaching fossil teeth of unknown age and to verifying life history profiles of human ancestors. This pilot study aims to (a) non-destructively image TCA in teeth from a known age archeological human population by propagation phase contrast X-ray synchrotron µCT (PPC-SR-µCT) (b) test the correlation between real and estimated ages, and the accuracy, precision and bias of age estimates. MATERIALS AND METHODS: We examine 20 permanent human canines (aged 20-81 years), from a 18th to 19th century known age collection from St. Luke's Church (London, England). We scanned transverse segments of acellular cementum in the apical portion of the middle root third using PPC-SR-µCT. We generated virtual transverse sections on which two observers perform two sessions of blind TCA counts. We calculate the estimated ages at death by adding 10 years to the TCA counts. RESULTS: A moderately strong positive linear relationship exists between real and estimated ages (r = 0.76, p < .001), with an average inaccuracy of 16.1 years and an average bias towards underestimation of 15.7 years. This difference is lower in individuals <50 years (6.8 and 6.5 years, respectively, n = 10) compared with those >50 years (24.9 years, n = 10). DISCUSSION: We reliably imaged and identified TCA in individuals <50 years from a known-age archeological sample. Scanning refinement will yield a promising alternative to current destructive methods of TCA analyses and to aid access to life history events in adult fossil hominins.

Indels, structural variation, and recombination drive genomic diversity in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum has a great capacity for evolutionary adaptation to evade host immunity and develop drug resistance. Current understanding of parasite evolution is impeded by the fact that a large fraction of the genome is either highly repetitive or highly variable and thus difficult to analyze using short-read sequencing technologies. Here, we describe a resource of deep sequencing data on parents and progeny from genetic crosses, which has enabled us to perform the first genome-wide, integrated analysis of SNP, indel and complex polymorphisms, using Mendelian error rates as an indicator of genotypic accuracy. These data reveal that indels are exceptionally abundant, being more common than SNPs and thus the dominant mode of polymorphism within the core genome. We use the high density of SNP and indel markers to analyze patterns of meiotic recombination, confirming a high rate of crossover events and providing the first estimates for the rate of non-crossover events and the length of conversion tracts. We observe several instances of meiotic recombination within copy number variants associated with drug resistance, demonstrating a mechanism whereby fitness costs associated with resistance mutations could be compensated and greater phenotypic plasticity could be acquired.

Analysis of protein-coding genetic variation in 60,706 humans.

Large-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) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This 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 have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having 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.

Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia.

We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination.

Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing.

Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss a methodology to significantly improve the success of P. vivax WGS on natural (non-adapted) patient isolates. Using 37 patient isolates from Indonesia, Thailand, and travellers, we assessed the application of CF11-based white blood cell filtration alone and in combination with short term ex vivo schizont maturation. Although CF11 filtration reduced human DNA contamination in 8 Indonesian isolates tested, additional short-term culture increased the P. vivax DNA yield from a median of 0.15 to 6.2 ng µl(-1) packed red blood cells (pRBCs) (p = 0.001) and reduced the human DNA percentage from a median of 33.9% to 6.22% (p = 0.008). Furthermore, post-CF11 and culture samples from Thailand gave a median P. vivax DNA yield of 2.34 ng µl(-1) pRBCs, and 2.65% human DNA. In 22 P. vivax patient isolates prepared with the 2-step method, we demonstrate high depth (median 654X coverage) and breadth (≥89%) of coverage on the Illumina GAII and HiSeq platforms. In contrast to the A+T-rich P. falciparum genome, negligible bias was observed in coverage depth between coding and non-coding regions of the P. vivax genome. This uniform coverage will greatly facilitate the detection of SNPs and copy number variants across the genome, enabling unbiased exploration of the natural diversity in P. vivax populations.

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

Comparison of eukaryotic phylogenetic profiling approaches using species tree aware methods.

BACKGROUND: Phylogenetic profiling encompasses an important set of methodologies for in silico high throughput inference of functional relationships between genes. The simplest profiles represent the distribution of gene presence-absence in a set of species as a sequence of 0's and 1's, and it is assumed that functionally related genes will have more similar profiles. The methodology has been successfully used in numerous studies of prokaryotic genomes, although its application in eukaryotes appears problematic, with reported low accuracy due to the complex genomic organization within this domain of life. Recently some groups have proposed an alternative approach based on the correlation of homologous gene group sizes, taking into account all potentially informative genetic events leading to a change in group size, regardless of whether they result in a de novo group gain or total gene group loss. RESULTS: We have compared the performance of classical presence-absence and group size based approaches using a large, diverse set of eukaryotic species. In contrast to most previous comparisons in Eukarya, we take into account the species phylogeny. We also compare the approaches using two different group categories, based on orthology and on domain-sharing. Our results confirm a limited overall performance of phylogenetic profiling in eukaryotes. Although group size based approaches initially showed an increase in performance for the domain-sharing based groups, this seems to be an overestimation due to a simplistic negative control dataset and the choice of null hypothesis rejection criteria. CONCLUSION: Presence-absence profiling represents a more accurate classifier of related versus non-related profile pairs, when the profiles under consideration have enough information content. Group size based approaches provide a complementary means of detecting domain or family level co-evolution between groups that may be elusive to presence-absence profiling. Moreover positive correlation between co-evolution scores and functional links imply that these methods could be used to estimate functional distances between gene groups and to cluster them based on their functional relatedness. This study should have important implications for the future development and application of phylogenetic profiling methods, not only in eukaryotic, but also in prokaryotic datasets.

Testing the neutral fixation of hetero-oligomerism in the archaeal chaperonin CCT.

The evolutionary transition from homo-oligomerism to hetero-oligomerism in multimeric proteins and its contribution to function innovation and organism complexity remain to be investigated. Here, we undertake the challenge of contributing to this theoretical ground by investigating the hetero-oligomerism in the molecular chaperonin cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) from archaea. CCT is amenable to this study because, in contrast to eukaryotic CCTs where sub-functionalization after gene duplication has been taken to completion, archaeal CCTs present no evidence for subunit functional specialization. Our analyses yield additional information to previous reports on archaeal CCT paralogy by identifying new duplication events. Analyses of selective constraints show that amino acid sites from 1 subunit have fixed slightly deleterious mutations at inter-subunit interfaces after gene duplication. These mutations have been followed by compensatory mutations in nearby regions of the same subunit and in the interface contact regions of its paralogous subunit. The strong selective constraints in these regions after speciation support the evolutionary entrapment of CCTs as hetero-oligomers. In addition, our results unveil different evolutionary dynamics depending on the degree of CCT hetero-oligomerism. Archaeal CCT protein complexes comprising 3 distinct classes of subunits present 2 evolutionary processes. First, slightly deleterious and compensatory mutations were fixed neutrally at inter-subunit regions. Second, sub-functionalization may have occurred at substrate-binding and adenosine triphosphate-binding regions after the 2nd gene duplication event took place. CCTs with 2 distinct types of subunits did not present evidence of sub-functionalization. Our results provide the 1st in silico evidence for the neutral fixation of hetero-oligomerism in archaeal CCTs and provide information on the evolution of hetero-oligomerism toward sub-functionalization in archaeal CCTs.

Artifactual phylogenies caused by correlated distribution of substitution rates among sites and lineages: the good, the bad, and the ugly.

Despite the advances in understanding molecular evolution, current phylogenetic methods barely take account of a fraction of the complexity of evolution. We are chiefly constrained by our incomplete knowledge of molecular evolutionary processes and the limits of computational power. These limitations lead to the establishment of either biologically simplistic models that rarely account for a fraction of the complexity involved or overfitting models that add little resolution to the problem. Such oversimplified models may lead us to assign high confidence to an incorrect tree (inconsistency). Rate-across-site (RAS) models are commonly used evolutionary models in phylogenetic studies. These account for heterogeneity in the evolutionary rates among sites but do not account for changing within-site rates across lineages (heterotachy). If heterotachy is common, using RAS models may lead to systematic errors in tree inference. In this work we show possible misleading effects in tree inference when the assumption of constant within-site rates across lineages is violated using maximum likelihood. Using a simulation study, we explore the ways in which gamma stationary models can lead to wrong topology or to deceptive bootstrap support values when the within-site rates change across lineages. More precisely, we show that different degrees of heterotachy mislead phylogenetic inference when the model assumed is stationary. Finally, we propose a geometry-based approach to visualize and to test for the possible existence of bias due to heterotachy.