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Moths and butterflies (Lepidoptera) have a heterogametic sex chromosome system with females carrying ZW chromosomes and males ZZ. The lack of W chromosomes in early diverging lepidopteran lineages has led to the suggestion of an ancestral Z0 system in this clade and a B chromosome origin of the W. This contrasts with the canonical model of W chromosome evolution in which the W would have originated from the same homologous autosomal pair as the Z chromosome. Despite the distinct models proposed, the rapid evolution of the W chromosome has hindered the elucidation of its origin. Here, we present high-quality, chromosome-level genome assemblies of twoHypolimnasspecies (Hypolimnas bolinaandHypolimnas misippus)and use theH. misippusassembly to explore the evolution of W chromosomes in butterflies and moths. We show that inH. misippusthe W chromosome has higher similarity to the Z chromosome than any other chromosome, which could suggest a possible origin from the same homologous autosome pair as the Z chromosome. However, using genome assemblies of closely related species (ditrysian lineages) containing assembled W chromosomes, we present contrasting evidence suggesting that the W chromosome might have evolved from a B chromosome instead. Crucially, by using a synteny analysis to infer homology, we show that W chromosomes are likely to share a common evolutionary origin in Lepidoptera. This study highlights the difficulty of studying the evolution of W chromosomes and contributes to better understanding its evolutionary origins.SignificanceButterflies and moths have a sex determination system in which females carry two different sex chromosomes, Z and W, while males carry two copies of the Z. The evolutionary origin of the W chromosome has been elusive, with many possible scenarios being suggested, such as the independent evolution of W chromosomes in many butterfly and moth species. Here, we present genome assemblies of twoHypolimnasbutterfly species and use one of them to shed light on the evolution of the W chromosome. We show that W chromosomes across butterflies and moths are very similar which suggests a shared common origin.

Sex chromosomes are evolutionarily labile in many animals, and fusion with an autosome is one of the mechanisms by which they can evolve. Sex chromosome-autosome (SA) fusions can reduce sexual conflict and promote adaptation and reproductive isolation among species. Recently, advances in genomics have fuelled the discovery of SA fusions in multiple vertebrates and in some invertebrates such as Lepidoptera. Here, we discovered multiple SA fusions in thesara/saphoclade of the classical adaptive radiation ofHeliconiusbutterflies. While the vast majority ofHeliconiusspecies have 21 chromosomes, species of the particularly rapidly diversifyingsaphoclade have up to 60 chromosomes. We found a sex chromosome fusion with chromosome 4 at the base of thesaphoclade and two additional sex chromosome fusions, each shared by two species. These sequential fusions between autosomes and sex chromosomes make theHeliconius saphoclade an ideal system to study the role of neo-sex chromosomes in adaptive radiations and the degeneration of sex chromosomes over time. Our study adds to a small but growing number of examples in butterflies with sex chromosome fusions that will help to unravel the importance of such rearrangements in the evolution of Lepidoptera and eukaryotes in general.Author SummaryChromosome number and structure are fundamental characteristics of a species. However, chromosomal rearrangements can occur spontaneously within a species, which can have significant consequences for recombination, segregation, speciation, and adaptation. Despite the importance of chromosomal rearrangements in evolution, we still have limited knowledge of the drivers and consequences of chromosomal fusions and fissions in natural populations, especially in adaptive radiations such asHeliconiusbutterflies. For the first time, we document chromosomal fusions between sex chromosomes and autosomes inHeliconius, particularly in species from thesara/saphoclade. Our research provides evidence for sex-autosome fusions involving autosomes 4, 9, and 14. All of these fusions seem to be associated with speciation events in this clade, with the sex-autosome 4 fusion being the oldest one. Although we do not yet understand the role or evolutionary consequences of these fusions, our study shows that chromosomal structure can evolve rapidly within a clade and generate chromosomal diversity. Overall, our findings contribute to a better understanding of chromosome evolution in highly diverse species.

It has become apparent from ancient DNA analysis, that the history of many human populations from across the globe are often complex, involving multiple population split, admixture, migration and isolation events. Local ancestry inference (LAI) aims to identify from which ancestral population chromosomal segments in admixed individuals are inherited. However, ancestry in existing LAI tools is characterised by a discrete population identity, a definition which is limited in the context of a complex demographic history involving multiple admixture events at different times. Moreover, many LAI tools rely on a reference panel of present day genomes that act as proxies for the ancestral populations. For ancient admixture events, these proxy genomes are likely only distantly related to the true ancestral populations. Here we present a new method that leverages advances in ancient DNA sequencing and genealogical inference to address these in issues in LAI. The method applies machine learning to tree sequences inferred for ancient and present day genomes and is based on a deterministic model of population structure, within which we introduce the concept of path ancestry. We show that the method is robust to a variety of demographic scenarios, generalises over model misspecification and that it outperforms a leading local ancestry inference tool. We further describe a downstream method to estimate the time since admixture for individuals with painted chromosomes. We apply the method to a large ancient DNA dataset covering Europe and West Eurasia and show that the inferred admixture ages are a better metric than sample ages alone for understanding movements of people across Europe in the past.

BackgroundPacBio high fidelity (HiFi) sequencing reads are both long (15-20 kb) and highly accurate (>Q20). Because of these properties, they have revolutionised genome assembly leading to more accurate and contiguous genomes. In eukaryotes the mitochondrial genome is sequenced alongside the nuclear genome often at very high coverage. A dedicated tool for mitochondrial genome assembly using HiFi reads is still missing.ResultsMitoHiFi was developed within the Darwin Tree of Life Project to assemble mitochondrial genomes from the HiFi reads generated for target species. The input for MitoHiFi is either the raw reads or the assembled contigs, and the tool outputs a mitochondrial genome sequence fasta file along with annotation of protein and RNA genes. Variants arising from heteroplasmy are assembled independently, and nuclear insertions of mitochondrial sequences are identified and not used in organellar genome assembly. MitoHiFi has been used to assemble 374 mitochondrial genomes (369 from 12 phyla and 39 orders of Metazoa and from 6 species of Fungi) for the Darwin Tree of Life Project, the Vertebrate Genomes Project and the Aquatic Symbiosis Genome Project. Inspection of 60 mitochondrial genomes assembled with MitoHiFi for species that already have reference sequences in public databases showed the widespread presence of previously unreported repeats.ConclusionsMitoHiFi is able to assemble mitochondrial genomes from a wide phylogenetic range of taxa from Pacbio HiFi data. MitoHiFi is written in python and is freely available on github (https://github.com/marcelauliano/MitoHiFi). MitoHiFi is available with its dependencies as a singularity image on github (ghcr.io/marcelauliano/mitohifi:master).

Summary The Eurasian Holocene (beginning c. 12 thousand years ago) encompassed some of the most significant changes in human evolution, with far-reaching consequences for the dietary, physical and mental health of present-day populations. Using an imputed dataset of >1600 complete ancient genome sequences, and new computational methods for locating selection in time and space, we reconstructed the selection landscape of the transition from hunting and gathering, to farming and pastoralism across West Eurasia. We identify major selection signals related to metabolism, possibly associated with the dietary shift occurring in this period. We show that the selection on loci such as the FADS cluster, associated with fatty acid metabolism, and the lactase persistence locus, began earlier than previously thought. A substantial amount of selection is also found in the HLA region and other loci associated with immunity, possibly due to the increased exposure to pathogens during the Neolithic, which may explain the current high prevalence of auto-immune disease, such as psoriasis, due to genetic trade-offs. By using ancient populations to infer local ancestry tracks in hundreds of thousands of samples from the UK Biobank, we find strong genetic differentiation among ancient Europeans in loci associated with anthropometric traits and susceptibility to several diseases that contribute to present-day disease burden. These were previously thought to be caused by local selection, but in fact can be attributed to differential genetic contributions from various source populations that are ancestral to present-day Europeans. Thus, alleles associated with increased height seem to have increased in frequency following the Yamnaya migration into northwestern Europe around 5,000 years ago. Alleles associated with increased risk of some mood-related phenotypes are overrepresented in the farmer ancestry component entering Europe from Anatolia around 11,000 years ago, while western hunter-gatherers show a strikingly high contribution of alleles conferring risk of traits related to diabetes. Our results paint a picture of the combined contributions of migration and selection in shaping the phenotypic landscape of present-day Europeans that suggests a combination of ancient selection and migration, rather than recent local selection, is the primary driver of present-day phenotypic differences in Europe.

Genome sequences are computationally assembled from millions of much shorter sequencing reads. Although this process can be impressively accurate with long reads, it is still subject to a variety of types of errors, including large structural misassembly errors in addition to localised base pair substitutions. Recent advances in long single molecule sequencing in combination with other long-range technologies such as synthetic long read clouds and Hi-C have dramatically increased the contiguity of assembly. This makes it all the more important to be able to validate the structural integrity of the chromosomal scale assemblies now being generated. Here we describe a novel assembly evaluation tool, Asset, which evaluates the consistency of a proposed genome assembly with multiple primary long-range data sets, identifying both supported regions and putative structural misassemblies. We present tests on three de novo assemblies from a human, a goat and a fish species, demonstrating that Asset can identify structural misassemblies accurately by combining regionally supported evidence from long read and other raw sequencing data. Not only can Asset be used to assess overall assembly confidence, and discover specific problematic regions for downstream genome curation, a process that leads to improvement in genome quality, but it can also provide feedback to automated assembly pipelines.

African cichlid fishes not only exhibit remarkably high rates of speciation but also have some of the fastest evolving sex determination systems in vertebrates. However, little is known empirically in cichlids about the genetic mechanisms generating new sex-determining variants, what forces dictate their fate, the demographic scales at which they evolve, and whether they are related to speciation. To address these questions, we looked for sex-associated loci in full genome data from 647 individuals of Astatotilapia calliptera from Lake Masoko, a small isolated crater lake in Tanzania, which contains two distinct ecomorphs of the species. We identified three separate XY systems on recombining chromosomes. Two Y alleles derive from mutations that increase expression of the gonadal soma-derived factor gene (gsdf) on chromosome 7; the first is a tandem duplication of the entire gene observed throughout much of the Lake Malawi haplochromine cichlid radiation to which A. calliptera belongs, and the second is a 5 kb insertion directly upstream of gsdf. Both the latter variant and another 700 bp insertion on chromosome 19 responsible for the third Y allele arose from transposable element insertions. Males belonging to the Masoko deep-water benthic ecomorph are determined exclusively by the gsdf duplication, whereas all three Y alleles are used in the Masoko littoral ecomorph, in which they appear to act antagonistically among males with different amounts of benthic admixture. This antagonism in the face of ongoing admixture may be important for sustaining multifactorial sex determination in Lake Masoko. In addition to identifying the molecular basis of three coexisting sex determining alleles, these results demonstrate that genetic interactions between Y alleles and genetic background can potentially affect fitness and adaptive evolution.

Epigenetic variation can alter transcription and promote phenotypic divergence between populations facing different environmental challenges. Here we assess the epigenetic basis of diversification during the early stages of speciation. We focus on the extent and functional relevance of DNA methylome divergence between two Astatotilapia calliptera ecomorphs in crater Lake Masoko, southern Tanzania. We report extensive genome-wide methylome divergence between populations linked to key biological processes, including transcriptional activity of ecologically-relevant genes. These include genes involved in steroid metabolism, haemoglobin composition and erythropoiesis, consistent with divergent habitat occupancy of the ecomorphs. Using a common garden experiment, we found that global methylation profiles are rapidly remodelled across generations, but ecomorph-specific differences can be inherited. Collectively, our study suggests an epigenetic contribution to early stages of vertebrate speciation.One sentence summaryInheritance and plasticity of epigenetic divergence characterise early stages of speciation in an incipient cichlid species of an African crater lake.

In 2001, Celera Genomics and the International Human Genome Sequencing Consortium published their initial drafts of the human genome, which revolutionized the field of genomics. While these drafts and the updates that followed effectively covered the euchromatic fraction of the genome, the heterochromatin and many other complex regions were left unfinished or erroneous. Addressing this remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium has finished the first truly complete 3.055 billion base pair (bp) sequence of a human genome, representing the largest improvement to the human reference genome since its initial release. The new T2T-CHM13 reference includes gapless assemblies for all 22 autosomes plus Chromosome X, corrects numerous errors, and introduces nearly 200 million bp of novel sequence containing 2,226 paralogous gene copies, 115 of which are predicted to be protein coding. The newly completed regions include all centromeric satellite arrays and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies for the first time.

Siamese fighting fish, commonly known as betta, are among the world’s most popular and morphologically diverse pet fish, but the genetic processes leading to their domestication and phenotypic diversification are largely unknown. We assembled de novo the genome of a wildBetta splendensand whole-genome sequenced multiple individuals across five species within theB. splendensspecies complex, including wild populations and domesticated ornamental betta. Given our estimate of the mutation rate from pedigrees, our analyses suggest that betta were domesticated at least 1,000 years ago, centuries earlier than previously thought. Ornamental betta individuals have variable contributions from otherBettaspecies and have also introgressed into wild populations of those species. We identifydmrt1as the main sex determination gene in ornamental betta but not in wildB. splendens, and find evidence for recent directional selection at the X-allele of the locus. Furthermore, we find genes with signatures of recent, strong selection that have large effects on color in specific parts of the body, or the shape of individual fins, and are almost all unlinked. Our results demonstrate how simple genetic architectures paired with anatomical modularity can lead to vast phenotypic diversity generated during animal domestication, and set the stage for using betta as a modern system for evolutionary genetics.One-Sentence SummaryGenomic analyses reveal betta fish were domesticated more than 1,000 years ago and the genes that changed in the process.

Many genome assemblies have been found to be incomplete and contain misassemblies. The Vertebrate Genomes Project (VGP) has been producing assemblies with an emphasis on being as complete and error-free as possible, utilizing long reads, long-range scaffolding data, new assembly algorithms, and manual curation. Here we evaluate these new vertebrate genome assemblies relative to the previous references for the same species, including a mammal (platypus), two birds (zebra finch, Anna’s hummingbird), and a fish (climbing perch). We found that 3 to 11% of genomic sequence was entirely missing in the previous reference assemblies, which included nearly entire GC-rich and repeat-rich microchromosomes with high gene density. Genome-wide, between 25 to 60% of the genes were either completely or partially missing in the previous assemblies, and this was in part due to a bias in GC-rich 5’-proximal promoters and 5’ exon regions. Our findings reveal novel regulatory landscapes and protein coding sequences that have been greatly underestimated in previous assemblies and are now present in the VGP assemblies.

SummaryHuman Induced Pluripotent Stem Cells (hiPSC) are an established patient-specific model system where opportunities are emerging for cell-based therapies. We contrast hiPSCs derived from different tissues, skin and blood, in the same individual. We show extensive single-nucleotide mutagenesis in all hiPSC lines, although fibroblast-derived hiPSCs (F-hiPSCs) are particularly heavily mutagenized by ultraviolet(UV)-related damage. We utilize genome sequencing data on 454 F-hiPSCs and 44 blood-derived hiPSCs (B-hiPSCs) to gain further insights. Across 324 whole genome sequenced(WGS) F-hiPSCs derived by the Human Induced Pluripotent Stem Cell Initiative (HipSci), UV-related damage is present in ~72% of cell lines, sometimes causing substantial mutagenesis (range 0.25-15 per Mb). Furthermore, we find remarkable genomic heterogeneity between independent F-hiPSC clones derived from the same reprogramming process in the same donor, due to oligoclonal populations within fibroblasts. Combining WGS and exome-sequencing data of 452 HipSci F-hiPSCs, we identify 272 predicted pathogenic mutations in cancer-related genes, of which 21 genes were hit recurrently three or more times, involving 77 (17%) lines. Notably, 151 of 272 mutations were present in starting fibroblast populations suggesting that more than half of putative driver events in F-hiPSCs were acquired in vivo. In contrast, B-hiPSCs reprogrammed from erythroblasts show lower levels of genome-wide mutations (range 0.28-1.4 per Mb), no UV damage, but a strikingly high prevalence of acquired BCOR mutations of ~57%, indicative of strong selection pressure. All hiPSCs had otherwise stable, diploid genomes on karyotypic pre-screening, highlighting how copy-number-based approaches do not have the required resolution to detect widespread nucleotide mutagenesis. This work strongly suggests that models for cell-based therapies require detailed nucleotide-resolution characterization prior to clinical application.

During the last decade, large volumes of ancient DNA (aDNA) data have been generated as part of whole-genome shotgun and target capture sequencing studies. This includes sequences from non-recombining loci such as the mitochondrial or Y chromosomes. However, given the highly degraded nature of aDNA data, post-mortem deamination and often low genomic coverage, combining ancient and modern samples for phylogenetic analyses remains difficult. Without care, these factors can lead to incorrect placement.For the Y chromosomes, current standard methods focus on curated markers, but these contain only a subset of the total variation. Examining all polymorphic markers is particularly important for low coverage aDNA data because it substantially increases the number of overlapping sites between present-day and ancient individuals which may lead to higher resolution phylogenetic placement. We provide an automated workflow for jointly analysing ancient and present-day sequence data in a phylogenetic context. For each ancient sample, we effectively evaluate the number of ancestral and derived alleles present on each branch and use this information to place ancient lineages to their most likely position in the phylogeny. We provide both a parsimony approach and a highly optimised likelihood-based approach that assigns a posterior probability to each branch.To illustrate the application of this method, we have compiled and make available the largest public Y-chromosomal dataset to date (2,014 samples) which we used as a reference for phylogenetic placement. We process publicly available African ancient DNA Y-chromosome sequences and examine how patterns of Y-chromosomal diversity change across time and the relationship between ancient and present-day lineages. The same software can be used to place samples with large amounts of missing data into other large non-recombining phylogenies such as the mitochondrial tree.

Epigenetic variation modulates gene expression and can be heritable. However, knowledge of the contribution of epigenetic divergence to adaptive diversification in nature remains limited. The massive evolutionary radiation of Lake Malawi cichlid fishes displaying extensive phenotypic diversity despite extremely low sequence divergence is an excellent system to study the epigenomic contribution to adaptation. Here, we present a comparative genome-wide methylome and transcriptome study, focussing on liver and muscle tissues in phenotypically divergent cichlid species. In both tissues we find substantial methylome divergence among species. Differentially methylated regions (DMR), enriched in evolutionary young transposons, are associated with transcription changes of ecologically-relevant genes related to energy expenditure and lipid metabolism, pointing to a link between dietary ecology and methylome divergence. Unexpectedly, half of all species-specific DMRs are shared across tissues and are enriched in developmental genes, likely reflecting distinct epigenetic developmental programmes. Our study reveals substantial methylome divergence in closely-related cichlid fishes and represents a resource to study the role of epigenetics in species diversification., The Lake Malawi cichlid fishes are an example of extreme vertebrate radiation; however, there is very little sequence divergence among the species. Here the authors present a comparative genome-wide methylome study to suggest DNA methylation played a major role in the extensive phenotypic diversity amongst these fishes.

Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly. As part of the Vertebrate Genomes Project (VGP) we have developed mitoVGP, a fully automated pipeline for similarity-based identification of mitochondrial reads and de novo assembly of mitochondrial genomes that incorporates both long (>10 kbp, PacBio or Nanopore) and short (100-300 bp, Illumina) reads. Our pipeline led to successful complete mitogenome assemblies of 100 vertebrate species of the VGP. We have observed that tissue type and library size selection have considerable impact on mitogenome sequencing and assembly. Comparing our assemblies to purportedly complete reference mitogenomes based on short-read sequencing, we have identified errors, missing sequences, and incomplete genes in those references, particularly in repeat regions. Our assemblies have also identified novel gene region duplications, shedding new light on mitochondrial genome evolution and organization.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are only available for a few non-microbial species1–4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling the most accurate and complete reference genomes to date. Here we summarize these developments, introduce a set of quality standards, and present lessons learned from sequencing and assembling 16 species representing major vertebrate lineages (mammals, birds, reptiles, amphibians, teleost fishes and cartilaginous fishes). We confirm that long-read sequencing technologies are essential for maximizing genome quality and that unresolved complex repeats and haplotype heterozygosity are major sources of error in assemblies. Our new assemblies identify and correct substantial errors in some of the best historical reference genomes. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an effort to generate high-quality, complete reference genomes for all ~70,000 extant vertebrate species and help enable a new era of discovery across the life sciences.

Background Diploid genome assembly is typically impeded by heterozygosity because it introduces errors when haplotypes are collapsed into a consensus sequence. Trio binning offers an innovative solution that exploits heterozygosity for assembly. Short, parental reads are used to assign parental origin to long reads from their F1 offspring before assembly, enabling complete haplotype resolution. Trio binning could therefore provide an effective strategy for assembling highly heterozygous genomes, which are traditionally problematic, such as insect genomes. This includes the wood tiger moth (Arctia plantaginis), which is an evolutionary study system for warning colour polymorphism. Findings We produced a high-quality, haplotype-resolved assembly for Arctia plantaginis through trio binning. We sequenced a same-species family (F1 heterozygosity ∼1.9%) and used parental Illumina reads to bin 99.98% of offspring Pacific Biosciences reads by parental origin, before assembling each haplotype separately and scaffolding with 10X linked reads. Both assemblies are contiguous (mean scaffold N50: 8.2 Mb) and complete (mean BUSCO completeness: 97.3%), with annotations and 31 chromosomes identified through karyotyping. We used the assembly to analyse genome-wide population structure and relationships between 40 wild resequenced individuals from 5 populations across Europe, revealing the Georgian population as the most genetically differentiated with the lowest genetic diversity. Conclusions We present the first invertebrate genome to be assembled via trio binning. This assembly is one of the highest quality genomes available for Lepidoptera, supporting trio binning as a potent strategy for assembling heterozygous genomes. Using our assembly, we provide genomic insights into the geographic population structure of A. plantaginis.

The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.

Motivation Rapid development in long read sequencing and scaffolding technologies is accelerating the production of reference-quality assemblies for large eukaryotic genomes. However, haplotype divergence in regions of high heterozygosity often results in assemblers creating two copies rather than one copy of a region, leading to breaks in contiguity and compromising downstream steps such as gene annotation. Several tools have been developed to resolve this problem. However, they either only focus on removing contained duplicate regions, also known as haplotigs, or fail to use all the relevant information and hence make errors. Results Here we present a novel tool “purge_dups” that uses sequence similarity and read depth to automatically identify and remove both haplotigs and heterozygous overlaps. In comparison with the current standard, purge_haplotigs, we demonstrate that purge_dups can reduce heterozygous duplication and increase assembly continuity while maintaining completeness of the primary assembly. Moreover, purge_dups is fully automatic and can be easy integrated into assembly pipelines. Availability The source code is written in C and is available at https://github.com/dfguan/purge_dups . Contact ydwang@hit.edu.cn , rd109@cam.ac.uk, This work was supported by the National Key Research and Development Program of China [2017YFC0907503, 2018YFC0910504 and 2017YFC1201201 to D.G. and Y.W.]; China Scholarship Council to D.G.; Wellcome Trust [WT207492 to S.A.M. and R.D., and WT206194 to J.W. and K.H.].

Methods to deconvolve single-cell RNA sequencing (scRNAseq) data are necessary for samples containing a natural mixture of genotypes and for scRNAseq experiments that multiplex cells from different donors1. Multiplexing across donors is a popular experimental design with many benefits including avoiding batch effects2, reducing costs, and improving doublet detection. Using variants detected in the RNAseq reads, it is possible to assign cells to the individuals from which they arose. These variants can also be used to identify and remove cross-genotype doublet cells that may have highly similar transcriptional profiles precluding detection by transcriptional profile. More subtle cross-genotype variant contamination can be used to estimate the amount of ambient RNA in the system. Ambient RNA is caused by cell lysis prior to droplet partitioning and is an important confounder of scRNAseq analysis3. Souporcell is a novel method to cluster cells using only the genetic variants detected within the scRNAseq reads. We show that it achieves high accuracy on genotype clustering, doublet detection, and ambient RNA estimation as demonstrated across a wide range of challenging scenarios.

Estimation of the relationship between DNA sequences is one of the most important problems in genomics. Understanding these relationships is central to demographic inference, correction of population structure in GWAS, identifying signals of selection etc. The data structure containing the full information about sample genealogy is called the ancestral recombination graph (ARG). However, ARG inference is a very difficult problem, not least due to a very complex state space. In this work we describe a new approach for fast and scalable generation of local tree topologies relating large numbers of haplotypes. Our method is closely related to the estimation of ARG, and captures both local and global properties of an ARG. It is based on a data structure which we call tree consistent PBWT, a modification of PBWT data structure introduced by R. Durbin (2014). We also explore some methods to estimate the quality of the generated tree topologies and to make inferences based on them. At the end we discuss a probabilistic model which could potentially lead to the estimation of ARG node times.

Far northeastern Siberia has been occupied by humans for more than 40 thousand years. Yet, owing to a scarcity of early archaeological sites and human remains, its population history and relationship to ancient and modern populations across Eurasia and the Americas are poorly understood. Here, we analyze 34 ancient genome sequences, including two from fragmented milk teeth found at the ~31.6 thousand-year-old (kya) Yana RHS site, the earliest and northernmost Pleistocene human remains found. These genomes reveal complex patterns of past population admixture and replacement events throughout northeastern Siberia, with evidence for at least three large-scale human migrations into the region. The first inhabitants, a previously unknown population of “Ancient North Siberians” (ANS), represented by Yana RHS, diverged ~38 kya from Western Eurasians, soon after the latter split from East Asians. Between 20 and 11 kya, the ANS population was largely replaced by peoples with ancestry related to present-day East Asians, giving rise to ancestral Native Americans and “Ancient Paleosiberians” (AP), represented by a 9.8 kya skeleton from Kolyma River. AP are closely related to the Siberian ancestors of Native Americans, and ancestral to contemporary communities such as Koryaks and Itelmen. Paleoclimatic modelling shows evidence for a refuge during the last glacial maximum (LGM) in southeastern Beringia, suggesting Beringia as a possible location for the admixture forming both ancestral Native Americans and AP. Between 11 and 4 kya, AP were in turn largely replaced by another group of peoples with ancestry from East Asia, the “Neosiberians” from which many contemporary Siberians derive. We detect gene flow events in both directions across the Bering Strait during this time, influencing the genetic composition of Inuit, as well as Na Dene-speaking Northern Native Americans, whose Siberian-related ancestry components is closely related to AP. Our analyses reveal that the population history of northeastern Siberia was highly dynamic throughout the Late Pleistocene and Holocene. The pattern observed in northeastern Siberia, with earlier, once widespread populations being replaced by distinct peoples, seems to have taken place across northern Eurasia, as far west as Scandinavia.

Large cohorts of human iPSCs from healthy donors are potentially a powerful tool for investigating the relationship between genetic variants and cellular phenotypes. Here we integrate high content imaging, gene expression and DNA sequence datasets for over 100 human iPSC lines to identify the genetic basis of inter-individual variability in cell behaviour. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we identified genes that correlated in expression with intrinsic (genetic) and extrinsic (ECM) factors. However, variation in mRNA levels could not account for outlier cell behaviour. Instead, we identified rare, deleterious SNVs in the coding sequence of genes involved in ECM adhesion that occurred in cell lines that were outliers for one or more phenotypes such as cell spreading. These also correlated with altered germ layer differentiation on micropatterned surfaces. Our study thus establishes a strategy for integrating genetic and cell biological measurements for high-throughput analysis.

Human populations out of Africa have experienced at least two bouts of introgression from archaic humans, from Neanderthals and Denisovans. In Papuans there is prior evidence of both these introgressions. Here we present a new approach to detect segments of individual genomes of archaic origin without using an archaic reference genome. The approach is based on a hidden Markov model that identifies genomic regions with a high density of single nucleotide variants (SNVs) not seen in unadmixed populations. We show using simulations that this provides a powerful approach to identifying segments of archaic introgression with a small rate of false detection. Furthermore our approach is able to accurately infer admixture proportions and divergence time of human and archaic populations.We apply the model to detect archaic introgression in 89 Papuans and show how the identified segments can be assigned to likely Neanderthal or Denisovan origin. We report more Denisovan admixture than previous studies and directly find a shift in size distribution of fragments of Neanderthal and Denisovan origin that is compatible with a difference in admixture time. Furthermore, we identify small amounts of Denisova ancestry in West Eurasians, South East Asians and South Asians.

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like−/− mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

Reference genomes guide our interpretation of DNA sequence data. However, conventional linear references are fundamentally limited in that they represent only one version of each locus, whereas the population may contain multiple variants. When the reference represents an individual’s genome poorly, it can impact read mapping and introduce bias. Variation graphs are bidirected DNA sequence graphs that compactly represent genetic variation, including large scale structural variation such as inversions and duplications.1 Equivalent structures are produced by de novo genome assemblers.2,3 Here we present vg, a toolkit of computational methods for creating, manipulating, and utilizing these structures as references at the scale of the human genome. vg provides an efficient approach to mapping reads onto arbitrary variation graphs using generalized compressed suffix arrays,4 with improved accuracy over alignment to a linear reference, creating data structures to support downstream variant calling and genotyping. These capabilities make using variation graphs as reference structures for DNA sequencing practical at the scale of vertebrate genomes, or at the topological complexity of new species assemblies.

There is increasing recognition that a single, monoploid reference genome is a poor universal reference structure for human genetics, because it represents only a tiny fraction of human variation. Adding this missing variation results in a structure that can be described as a mathematical graph: a genome graph. We demonstrate that, in comparison to the existing reference genome (GRCh38), genome graphs can substantially improve the fractions of reads that map uniquely and perfectly. Furthermore, we show that this fundamental simplification of read mapping transforms the variant calling problem from one in which many non-reference variants must be discovered de-novo to one in which the vast majority of variants are simply re-identified within the graph. Using standard benchmarks as well as a novel reference-free evaluation, we show that a simplistic variant calling procedure on a genome graph can already call variants at least as well as, and in many cases better than, a state-of-the-art method on the linear human reference genome. We anticipate that graph-based references will supplant linear references in humans and in other applications where cohorts of sequenced individuals are available.

Mechanisms that suppress recombination are known to help maintain species barriers by preventing the breakup of co-adapted gene combinations. The sympatric butterfly speciesH. melpomeneandH. cydnoare separated by many strong barriers, but the species still hybridise infrequently in the wild, with around 40% of the genome influenced by introgression. We tested the hypothesis that genetic barriers between the species are reinforced by inversions or other mechanisms to reduce between-species recombination rate. We constructed fine-scale recombination maps for Panamanian populations of both species and hybrids to directly measure recombination rate between these species, and generated long sequence reads to detect inversions. We find no evidence for a systematic reduction in recombination rates in F1 hybrids, and also no evidence for inversions longer than 50 kb that might be involved in generating or maintaining species barriers. This suggests that mechanisms leading to global or local reduction in recombination do not play a significant role in the maintenance of species barriers betweenH. melpomeneandH. cydno.Author SummaryIt is now possible to study the process of species formation by sequencing the genomes of multiple closely related species.Heliconius melpomeneandHeliconius cydnoare two butterfly species that have diverged over the past 2 million years and have different colour patterns, mate preferences and host plants. However, they still hybridise infrequently in the wild and exchange large parts of their genomes. Typically, when genomes are exchanged, chromosomes are recombined and gene combinations are broken up, preventing species from forming. Theory predicts that gene variants that define species might be linked together because of structural differences in their genomes, such as inverted pieces of chromosomes that will not be broken up when the species hybridise. However, in this paper, we use deep sequencing of large crosses of butterflies to show that there are no long chromosome regions that are not broken up during hybridisation, and no long chromosome inversions anywhere between the two genomes. This suggests that hybridisation is rare enough and mate preference is strong enough that inversions are not necessary to maintain the species barrier.

Previous studies1,2 have shown that large multi-population imputation reference panels increases the number of well-imputed variants. However, to our knowledge, no previous studies have evaluated the rate of introduced variation in monomorphic sites of the study population when using imputation panels with admixed populations. In this study we evaluate the rate of false positive variants introduced by the imputation of Finnish genotype data using global reference panels (Haplotype Reference Consortium1; HRC, and the 1000Genomes project Phase I3; 1000G) and compare the results to a Finnish population-specific reference panel combining whole genome and exome sequenced samples. In sites that were monomorphic in our test set, we observed high false positive rates for the global reference panels (4.0% for 1000G and 2.6% for HRC) compared to the Finnish panel (0.26%). This rate was even higher (7.4%) when using a combination panel of 1000G and Finnish whole genome sequences with cross-panel imputation.

Structural rearrangements have long been recognized as an important source of genetic variation with implications in phenotypic diversity and disease, yet their evolutionary dynamics are difficult to characterize with short-read sequencing. Here, we report long-read sequencing for 12 strains representing major subpopulations of the partially domesticated yeastSaccharomyces cerevisiaeand its wild relativeSaccharomyces paradoxus. Complete genome assemblies and annotations generate population-level reference genomes and allow for the first explicit definition of chromosome partitioning into cores, subtelomeres and chromosome-ends. High-resolution view of structural dynamics uncovers that, in chromosomal cores,S. paradoxusexhibits higher accumulation rate of balanced structural rearrangements (inversions, translocations and transpositions) whereasS. cerevisiaeaccumulates unbalanced rearrangements (large insertions, deletions and duplications) more rapidly. In subtelomeres, recurrent interchromosomal reshuffling was found in both species, with higher rate inS. cerevisiae. Such striking contrasts between wild and domesticated yeasts reveal the influence of human activities on structural genome evolution.

The human reference genome assembly plays a central role in nearly all aspects of today’s basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009 and reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that while the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.

Isolated populations with enrichment of variants due to recent population bottlenecks provide a powerful resource for identifying disease-associated genetic variants and genes. As a model of an isolate population, we sequenced the genomes of 1463 Finnish individuals as part of the Sequencing Initiative Suomi (SISu) Project. We compared the genomic profiles of the 1463 Finns to a sample of 1463 British individuals that were sequenced in parallel as part of the UK10K Project. Whereas there were no major differences in the allele frequency of common variants, a significant depletion of variants in the rare frequency spectrum was observed in Finns when comparing the two populations. On the other hand, we observed >2.1 million variants that were twice as frequent among Finns compared to Britons and 800,000 variants that were more than 10 times more frequent in Finns. Furthermore, in Finns we observed a relative proportional enrichment of variants in the minor allele frequency range between 2 - 5% (p < 2.2×10−16). When stratified by their functional annotations, loss-of-function (LoF) variants showed the highest proportional enrichment in Finns (p = 0.0291). In the noncoding part of the genome, variants in conserved regions (p = 0.002) and promoters (p = 0.01) were also significantly enriched in the Finnish samples. These functional categories represent the highest a priori power for downstream association studies of rare variants using population isolates.

We are rapidly approaching the point where we have sequenced millions of human genomes. There is a pressing need for new data structures to store raw sequencing data and efficient algorithms for population scale analysis. Current reference based data formats do not fully exploit the redundancy in population sequencing nor take advantage of shared genetic variation. In recent years, the Burrows-Wheeler transform (BWT) and FM-index have been widely employed as a full text searchable index for read alignment and de novo assembly. We introduce the concept of a population BWT and use it to store and index the sequencing reads of 2,705 samples from the 1000 Genomes Project. A key feature is that as more genomes are added, identical read sequences are increasingly observed and compression becomes more efficient. We assess the support in the 1000 Genomes read data for every base position of two human reference assembly versions, identifying that 3.2 Mbp with population support was lost in the transition from GRCh37 with 13.7 Mbp added to GRCh38. We show that the vast majority of variant alleles can be uniquely described by overlapping 31-mers and show how rapid and accurate SNP and indel genotyping can be carried out across the genomes in the population BWT. We use the population BWT to carry out non-reference queries to search for the presence of all known viral genomes, and discover human T-lymphotropic virus 1 integrations in six samples in a recognised epidemiological distribution.

Heterozygous mutations within homozygous sequences descended from a recent common ancestor offer a way to ascertain de novo mutations (DNMs) across multiple generations. Using exome sequences from 3,222 British-Pakistani individuals with high parental relatedness, we estimate a mutation rate of 1. 45 ± 0.05 × 10−8 per base pair per generation in autosomal coding sequence, with a corresponding noncrossover gene conversion rate of 8.75 ± 0.05 × 10−6 per base pair per generation. This is at the lower end of exome mutation rates previously estimated in parent-offspring trios, suggesting that post-zygotic mutations contribute little to the human germline mutation rate. We found frequent recurrence of mutations at polymorphic CpG sites, and an increase in C to T mutations in a 5’ CCG 3’ → 5’ CTG 3’ context in the Pakistani population compared to Europeans, suggesting that mutational processes have evolved rapidly between human populations.

Induced pluripotent stem cell (iPSC) technology has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterisation of many existing iPSC lines limits their potential use for research and therapy. Here, we describe the systematic generation, genotyping and phenotyping of 522 open access human iPSCs derived from 189 healthy male and female individuals as part of the Human Induced Pluripotent Stem Cells Initiative (HipSci:http://www.hipsci.org). Our study provides a comprehensive picture of the major sources of genetic and phenotypic variation in iPSCs and establishes their suitability for use in genetic studies of complex human traits and cancer. Using a combination of genomewide analyses we find that 5-25% of the variation in different iPSC phenotypes, including differentiation capacity and cellular morphology, arises from differences betweenindividuals. We also assess the phenotypic effects of rare, genomic copy number mutations that are recurrently seen following iPSC reprogramming and present an initial map of common regulatory variants affecting the transcriptome of pluripotent cells in humans.

MotivationMost DNA sequence in diploid organisms is found in two copies, one contributed by the mother and the other by the father. The high density of differences between the maternally and paternally contributed sequences (heterozygous sites) in some organisms makes de novo genome assembly very challenging, even for algorithms specifically designed to deal with these cases. Therefore, various approaches, most commonly inbreeding in the laboratory, are used to reduce heterozygosity in genomic data prior to assembly. However, many species are not amenable to these techniques.ResultsWe introduce trio-sga, a set of three algorithms designed to take advantage of mother-father-offspring trio sequencing to facilitate better quality genome assembly in organisms with moderate to high levels of heterozygosity. Two of the algorithms use haplotype phase information present in the trio data to eliminate the majority of heterozygous sites before the assembly commences. The third algorithm is designed to reduce sequencing costs by enabling the use of parents’ reads in the assembly of the genome of the offspring. We test these algorithms on a ‘simulated trio’ from four hap-loid datasets, and further demonstrate their performance by assembling three highly heterozygous Heliconius butterfly genomes. While the implementation of trio-sga is tuned towards Illumina-generated data, we note that the trio approach to reducing heterozygosity is likely to have cross-platform utility for de novo assembly.

We describe a reference panel of 64,976 human haplotypes at 39,235,157 SNPs constructed using whole genome sequence data from 20 studies of predominantly European ancestry. Using this resource leads to accurate genotype imputation at minor allele frequencies as low as 0.1%, a large increase in the number of SNPs tested in association studies and can help to discover and refine causal loci. We describe remote server resources that allow researchers to carry out imputation and phasing consistently and efficiently.

De novo genome sequence assembly is important both to generate new sequence assemblies for previously uncharacterized genomes and to identify the genome sequence of individuals in a reference-unbiased way. We present memory efficient data structures and algorithms for assembly using the FM-index derived from the compressed Burrows-Wheeler transform, and a new assembler based on these called SGA (String Graph Assembler). We describe algorithms to error-correct, assemble, and scaffold large sets of sequence data. SGA uses the overlap-based string graph model of assembly, unlike most de novo assemblers that rely on de Bruijn graphs, and is simply parallelizable. We demonstrate the error correction and assembly performance of SGA on 1.2 billion sequence reads from a human genome, which we are able to assemble using 54 GB of memory. The resulting contigs are highly accurate and contiguous, while covering 95% of the reference genome (excluding contigs

The use of phylogenetic trees to describe the evolution of biological processes was established in the 1950s (Hennig 1952) and remains a fundamental approach to understanding the evolution of individual genes through to complete genomes; for example, in the mouse (Mouse Genome Sequencing Consortium 2002), rat (Gibbs et al. 2004), chicken (International Chicken Genome Sequencing Consortium 2004), and monodelphis (Mikkelsen et al. 2007) genome papers, and numerous papers on individual sequences. Now routine, the determination of vertebrate genome sequences provides a rich data source to understand evolution, and using phylogenetic trees of the genes is one of the best ways to organize these data. However, the increased set of genomes makes the compute and engineering tasks to form all the gene trees progressively more complex and harder for individual groups to use. The Ensembl project provides an accurate and consistent protein-coding gene set for all vertebrate genomes (International Human Genome Sequencing Consortium 2001; Dehal et al. 2002; Mouse Genome Sequencing Consortium 2002; Gibbs et al. 2004; Xie et al. 2005; Mikkelsen et al. 2007; Rhesus Macaque Genome Sequencing and Analysis Consortium 2007). Previously (until April 2006), Ensembl provided a basic method for tracing orthologs via the Best Reciprocal BLAST method, similar to approaches used in other genome analyses, such as Drosophila melanogaster (Adams et al. 2000) or human (International Human Genome Sequencing Consortium 2001). In June 2006 (Hubbard et al. 2007), we replaced this system with a phylogenetically sound, gene tree-based approach, providing a complete set of phylogenetic trees spanning 91% of genes across vertebrates. In addition to the vertebrates we have included a few important non-vertebrate species (fly, worm, and yeast) to act both as out groups and provide links to these model organisms. In this paper we provide the motivation, implementation, and benchmarking of this method and document the display and access methods for these trees. There have been a number of methods proposed for routine generation of genomewide orthology descriptions, including Inparanoid (Remm et al. 2001), MSOAR (Fu et al. 2007), OrthoMCL (Li et al. 2003), HomoloGene (Wheeler et al. 2008), TreeFam (Li et al. 2006), PhyOP (Goodstadt and Ponting 2006), and PhiGs (Dehal and Boore 2006). The first four, Inparanoid, MSOAR, OrthoMCL, and HomoloGene, focus on providing clusters (or linked clusters) of genes, without an explicit tree topology. PhyOP (Goodstadt and Ponting 2006) uses a tree-based method, but between pairs of closely related species, resolving paralogs accurately by using neutral substitution (as measured by d S, the synonymous substitution rate). TreeFam provides an explicit gene tree across multiple species, using both d S, d N (nonsynonymous substitution rate), nucleotide and protein distance measures, and the standard species tree to balance duplications vs. deletions to inform the tree construction, using the program TreeBeST (http://treesoft.sourceforge.net/treebest.shtml; L. Heng, A.J. Vilella, E. Birney, and R. Durbin, in prep.). The PhiGs method (Dehal and Boore 2006) is a leading phylogenetic-based method that produced a comprehensive phylogenetic resource for the genomes at the time it was run, and the basic outline of its analysis, which was clustering of protein sequences, followed by phylogenetic trees, is similar to the method presented here. However, the PhiGs resource covered a smaller number of species (23 vs. 45) and has been difficult to keep up to date with the advances in gene sets and genomes. Another major difference between PhiG-based phylogenetic trees and the phylogenetic trees presented here is that the former was calculated using a single maximum likelihood method based on protein evolution. In contrast, the Ensembl gene trees are calculated using a new method, TreeBeST, which integrates multiple tree topologies, in particular both DNA level and protein level models and combines this with a species-tree aware penalization of topologies, which are inconsistent with known species relationships. We show in this paper that this method produces trees that are more consistent with synteny relationships and less anomalous topologies than single protein-based phylogenetic methods. There are also many single phylogenetic tree-building approaches, many of them based on maximum likelihood methods; one leading method is PhyML (Guindon and Gascuel 2003). It is unclear what is the best method to use, in particular in the context of genome-wide tree building with constraints on computational costs and the need to robustly handle many complex scenarios usually involving large families with heterogeneous phylogenetic depths. In this paper, we benchmark in vertebrates the tree programs TreeBeST and PhyML, and the resulting trees to basic best reciprocal hit (BRH) methods, and cluster frameworks, in particular Inparanoid and HomoloGene. We also benchmark to a recent PhyOP data set. The PhyOP pipeline has recently switched to use the same tree-building program (TreeBeST) that we use, but differs in its input clusters. Although we adopted this same tree-building method, we describe here considerable novel engineering in the deployment of these methods across all vertebrates. Similar to the PhiGs resource, we have used the dense coverage of genomes to provide topologically based timings (i.e., the standard use of outgroups vs. subsequent lineages to bracket a duplication), in order to label duplication events.

New sequencing technologies promise a new era in the use of DNA sequence. However, some of these technologies produce very short reads, typically of a few tens of base pairs, and to use these reads effectively requires new algorithms and software. In particular, there is a major issue in efficiently aligning short reads to a reference genome and handling ambiguity or lack of accuracy in this alignment. Here we introduce the concept of mapping quality, a measure of the confidence that a read actually comes from the position it is aligned to by the mapping algorithm. We describe the software MAQ that can build assemblies by mapping shotgun short reads to a reference genome, using quality scores to derive genotype calls of the consensus sequence of a diploid genome, e.g., from a human sample. MAQ makes full use of mate-pair information and estimates the error probability of each read alignment. Error probabilities are also derived for the final genotype calls, using a Bayesian statistical model that incorporates the mapping qualities, error probabilities from the raw sequence quality scores, sampling of the two haplotypes, and an empirical model for correlated errors at a site. Both read mapping and genotype calling are evaluated on simulated data and real data. MAQ is accurate, efficient, versatile, and user-friendly. It is freely available at http://maq.sourceforge.net.

Complete gene knockouts are highly informative about gene function. We exome sequenced 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous likely loss of function (rhLOF) genotypes predicted to disrupt (knockout) 781 genes. Based on depletion of rhLOF genotypes, we estimate that 13.6% of knockouts are incompatible with adult life, finding on average 1.6 heterozygous recessive lethal LOF variants per adult. Linking to lifelong health records, we observed no association of rhLOF genotypes with prescription- or doctor-consultation rate, and no disease-related phenotypes in 33 of 42 individuals with rhLOF genotypes in recessive Mendelian disease genes. Phased genome sequencing of a healthy PRDM9 knockout mother, her child and controls, showed meiotic recombination sites localised away from PRDM9-dependent hotspots, demonstrating PRDM9 redundancy in humans.

Purging through inbreeding occurs when consanguineous marriages increases the rate at which deleterious alleles are present in a homozygous state. In this study we carried out low-read depth (4-10x) whole-genome sequencing in 568 individuals from three Italian founder populations, and compared it to data from other Italian and European populations from the 1000 Genomes Project. We show extended consanguinity and depletion of homozygous genotypes at potentially detrimental sites in the founder populations compared to outbred populations. However these patterns are not compatible with the hypothesis of consanguinity driving the purging of highly deleterious mutations according to simulations. Therefore we conclude that genetic drift and the founder effect should be responsible for the observed purging of deleterious variants.

Statistical factor analysis methods have previously been used to remove noise components from high dimensional data prior to genetic association mapping, and in a guided fashion to summarise biologically relevant sources of variation. Here we show how the derived factors summarising pathway expression can be used to analyse the relationships between expression, heritability and ageing. We used skin gene expression data from 647 twins from the MuTHER Consortium and applied factor analysis to concisely summarise patterns of gene expression, both to remove broad confounding influences and to produce concise pathway-level phenotypes. We derived 930 "pathway phenotypes" which summarised patterns of variation across 186 KEGG pathways (five phenotypes per pathway). We identified 69 significant associations of age with phenotype from 57 distinct KEGG pathways at a stringent Bonferroni threshold (P

The 3′ untranslated regions (3′ UTR) of eukaryotic genes can contain motifs involved in regulation of gene expression or localization at the post-transcriptional level. This study concerns the identification of novel, conserved elements in 3′ UTRs of many ribosomal protein mRNAs in Caenorhabditis elegans and Caenorhabditis briggsae. Analysis of the region around the polyadenylation signal in many ribosomal protein mRNAs indicates the conservation of a sequence motif UUGUU occurring both before and immediately after the polyadenylation signal. Building a statistical model of this motif and searching a database of C. elegans 3′ UTRs reveals that this motif is also present in the 3′ UTR of some genes involved in translation and ribosome maturation, among others. We suggest that this signal may be involved in translation or other message-level regulation of ribosomal genes in C. elegans.

We describe a method (implemented in a program, GAZE) for assembling arbitrary evidence for individual gene components (features) into predictions of complete gene structures. Our system is generic in that both the features themselves, and the model of gene structure against which potential assemblies are validated and scored, are external to the system and supplied by the user. GAZE uses a dynamic programming algorithm to obtain the highest scoring gene structure according to the model and posterior probabilities that each input feature is part of a gene. A novel pruning strategy ensures that the algorithm has a run-time effectively linear in sequence length. To demonstrate the flexibility of our system in the incorporation of additional evidence into the gene prediction process, we show how it can be used to both represent nonstandard gene structures (in the form oftrans-spliced genes in Caenorhabditis elegans), and make use of similarity information (in the form of Expressed Sequence Tag alignments), while requiring no change to the underlying software. GAZE is available athttp://www.sanger.ac.uk/Software/analysis/GAZE.

Understanding the genetic architecture of gene expression is an intermediate step to understand the genetic architecture of complex diseases. RNA-seq technologies have improved the quantification of gene expression and allow to measure allelic specific expression (ASE)1-3. ASE is hypothesized to result from the direct effect of cis regulatory variants, but a proper estimation of the causes of ASE has not been performed to date. In this study we take advantage of a sample of twins to measure the relative contribution of genetic and environmental effects on ASE and we found substantial effects of gene x gene (GxG) and gene x environment (GxE) interactions. We propose a model where ASE requires genetic variability in cis, a difference in the sequence of both alleles, but the magnitude of the ASE effect depends on trans genetic and environmental factors that interact with the cis genetic variants. We uncover large GxG and GxE effects on gene expression and likely complex phenotypes that currently remain elusive.

The availability of complete human genome sequences from populations across the world has given rise to new population genetic inference methods that explicitly model their ancestral relationship under recombination and mutation. So far, application of these methods to evolutionary history more recent than 20-30 thousand years ago and to population separations has been limited. Here we present a new method that overcomes these shortcomings. The Multiple Sequentially Markovian Coalescent (MSMC) analyses the observed pattern of mutations in multiple individuals, focusing on the first coalescence between any two individuals. Results from applying MSMC to genome sequences from nine populations across the world suggest that the genetic separation of non-African ancestors from African Yoruban ancestors started long before 50,000 years ago, and give information about human population history as recently as 2,000 years ago, including the bottleneck in the peopling of the Americas, and separations within Africa, East Asia and Europe.

Large-scale genomic sequencing requires a software infrastructure to support and integrate applications that are not directly compatible. We describe a suite of software tools built around the Common Assembly Format (CAF), a comprehensive representation of a sequence assembly as a text file. These tools form the backbone of sequencing informatics at the Sanger Centre and the Genome Sequencing Center. The CAF format is intentionally flexible, and our Perl and C libraries, which parse and manipulate it, provide powerful tools for creating new applications as well as wrappers to incorporate other software. The tools are available free by anonymous FTP from ftp://ftp.sanger.ac.uk/pub/badger/.

The GeneWise method for combining gene prediction and homology searches was applied to the 2.9-Mb region from Drosophila melanogaster. The results from the Genome Annotation Assessment Project (GASP) showed that GeneWise provided reasonably accurate gene predictions. Further investigation indicates that many of the incorrect gene predictions from GeneWise were due to transposons with valid protein-coding genes and the remaining cases are pseudogenes or possible annotation oversights.