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Several large-scale Illumina whole-genome sequencing (WGS) and whole-exome sequencing (WES) projects have emerged recently that have provided exceptional opportunities to discover mobile element insertions (MEIs) and study the impact of these MEIs on human genomes. However, these projects also have presented major challenges with respect to the scalability and computational costs associated with performing MEI discovery on tens or even hundreds of thousands of samples. To meet these challenges, we have developed a more efficient and scalable version of our mobile element locator tool (MELT) called CloudMELT. We then used MELT and CloudMELT to perform MEI discovery in 57,919 human genomes and exomes, leading to the discovery of 104,350 nonredundant MEIs. We leveraged this collection (1) to examine potentially active L1 source elements that drive the mobilization of new Alu, L1, and SVA MEIs in humans; (2) to examine the population distributions and subfamilies of these MEIs; and (3) to examine the mutagenesis of GENCODE genes, ENCODE-annotated features, and disease genes by these MEIs. Our study provides new insights on the L1 source elements that drive MEI mutagenesis and brings forth a better understanding of how this mutagenesis impacts human genomes.

Virtually all genome sequencing efforts in national biobanks, complex and Mendelian disease programs, and medical genetic initiatives are reliant upon short-read whole-genome sequencing (srWGS), which presents challenges for the detection of structural variants (SVs) relative to emerging long-read WGS (lrWGS) technologies. Given this ubiquity of srWGS in large-scale genomics initiatives, we sought to establish expectations for routine SV detection from this data type by comparison with lrWGS assembly, as well as to quantify the genomic properties and added value of SVs uniquely accessible to each technology. Analyses from the Human Genome Structural Variation Consortium (HGSVC) of three families captured ~11,000 SVs per genome from srWGS and ~25,000 SVs per genome from lrWGS assembly. Detection power and precision for SV discovery varied dramatically by genomic context and variant class: 9.7% of the current GRCh38 reference is defined by segmental duplication (SD) and simple repeat (SR), yet 91.4% of deletions that were specifically discovered by lrWGS localized to these regions. Across the remaining 90.3% of reference sequence, we observed extremely high (93.8%) concordance between technologies for deletions in these datasets. In contrast, lrWGS was superior for detection of insertions across all genomic contexts. Given that non-SD/SR sequences encompass 95.9% of currently annotated disease-associated exons, improved sensitivity from lrWGS to discover novel pathogenic deletions in these currently interpretable genomic regions is likely to be incremental. However, these analyses highlight the considerable added value of assembly-based lrWGS to create new catalogs of insertions and transposable elements, as well as disease-associated repeat expansions in genomic sequences that were previously recalcitrant to routine assessment.

Somatic LINE-1 (L1) retrotransposition has been detected in early embryos, adult brains, and the gastrointestinal (GI) tract, and many cancers, including epithelial GI tumors. We previously found numerous somatic L1 insertions in paired normal and GI cancerous tissues. Here, using a modified method of single-cell analysis for somatic L1 insertions, we studied adenocarcinomas of colon, pancreas, and stomach, and found a variable number of somatic L1 insertions in tumors of the same type from patient to patient. We detected no somatic L1 insertions in single cells of 5 of 10 tumors studied. In three tumors, aneuploid cells were detected by FACS. In one pancreatic tumor, there were many more L1 insertions in aneuploid than in euploid tumor cells. In one gastric cancer, both aneuploid and euploid cells contained large numbers of likely clonal insertions. However, in a second gastric cancer with aneuploid cells, no somatic L1 insertions were found. We suggest that when the cellular environment is favorable to retrotransposition, aneuploidy predisposes tumor cells to L1 insertions, and retrotransposition may occur at the transition from euploidy to aneuploidy. Seventeen percent of insertions were also present in normal cells, similar to findings in genomic DNA from normal tissues of GI tumor patients. We provide evidence that: 1) The number of L1 insertions in tumors of the same type is highly variable, 2) most somatic L1 insertions in GI cancer tissues are absent from normal tissues, and 3) under certain conditions, somatic L1 retrotransposition exhibits a propensity for occurring in aneuploid cells.

The 1000 Genomes Project (1kGP) is the largest fully open resource of whole-genome sequencing (WGS) data consented for public distribution without access or use restrictions. The final, phase 3 release of the 1kGP included 2,504 unrelated samples from 26 populations and was based primarily on low-coverage WGS. Here, we present a high-coverage 3,202-sample WGS 1kGP resource, which now includes 602 complete trios, sequenced to a depth of 30X using Illumina. We performed single-nucleotide variant (SNV) and short insertion and deletion (INDEL) discovery and generated a comprehensive set of structural variants (SVs) by integrating multiple analytic methods through a machine learning model. We show gains in sensitivity and precision of variant calls compared to phase 3, especially among rare SNVs as well as INDELs and SVs spanning frequency spectrum. We also generated an improved reference imputation panel, making variants discovered here accessible for association studies.

Complex structural variants (CSVs) are genomic alterations that have more than two breakpoints and are considered as simultaneous occurrence of simple structural variants. However, detecting the compounded mutational signals of CSVs is challenging through a commonly used model-match strategy. As a result, there has been limited progress for CSV discovery compared with simple structural variants. We systematically analyzed the multi-breakpoint connection feature of CSVs, and proposed Mako, utilizing a bottom-up guided model-free strategy, to detect CSVs from paired-end short-read sequencing. Specifically, we implemented a graph-based pattern growth approach, where the graph depicts potential breakpoint connections and pattern growth enables CSV detection without predefined models. Comprehensive evaluations on both simulated and real datasets revealed that Mako outperformed other algorithms. Notably, validation rates of CSV on real data based on experimental and computational validations as well as manual inspections are around 70%, where the medians of experimental and computational breakpoint shift are 13bp and 26bp, respectively. Moreover, Mako CSV subgraph effectively characterized the breakpoint connections of a CSV event and uncovered a total of 15 CSV types, including two novel types of adjacent segments swap and tandem dispersed duplication. Further analysis of these CSVs also revealed impact of sequence homology in the formation of CSVs. Mako is publicly available at https://github.com/jiadong324/Mako.

Human genomes are typically assembled as consensus sequences that lack information on parental haplotypes. Here we describe a reference-free workflow for diploid de novo genome assembly that combines the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing1,2 with continuous long-read or high-fidelity3 sequencing data. Employing this strategy, we produced a completely phased de novo genome assembly for each haplotype of an individual of Puerto Rican descent (HG00733) in the absence of parental data. The assemblies are accurate (quality value > 40) and highly contiguous (contig N50 > 23 Mbp) with low switch error rates (0.17%), providing fully phased single-nucleotide variants, indels and structural variants. A comparison of Oxford Nanopore Technologies and Pacific Biosciences phased assemblies identified 154 regions that are preferential sites of contig breaks, irrespective of sequencing technology or phasing algorithms., Assembly of haplotype-resolved human genomes is achieved by combining short and long reads.

Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent–child trio data. We present 64 assembled haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average contig N50: 26 Mbp) integrate all forms of genetic variation across even complex loci such as the major histocompatibility complex. We focus on 107,590 structural variants (SVs), of which 68% are inaccessible by short-read sequencing. We identify new SV hotspots (spanning megabases of gene-rich sequence), characterize 130 of the most active mobile element source elements, and find that 63% of all SVs arise by homology-mediated mechanisms—a twofold increase from previous studies. Our resource now enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1,525 expression quantitative trait loci (SV-eQTLs) as well as SV candidates for adaptive selection within the human population.

Resolving genomic structural variationMany human genomes have been reported using short-read technology, but it is difficult to resolve structural variants (SVs) using these data. These genomes thus lack comprehensive comparisons among individuals and populations. Ebertet al.used long-read structural variation calling across 64 human genomes representing diverse populations and developed new methods for variant discovery. This approach allowed the authors to increase the number of confirmed SVs and to describe the patterns of variation across populations. From this dataset, they identified quantitative trait loci affected by these SVs and determined how they may affect gene expression and potentially explain genome-wide association study hits. This information provides insights into patterns of normal human genetic variation and generates reference genomes that better represent the diversity of our species.Science, this issue p.eabf7117

Virtually all genome sequencing efforts in national biobanks, complex and Mendelian disease programs, and emerging clinical diagnostic approaches utilize short-reads (srWGS), which present constraints for genome-wide discovery of structural variants (SVs). Alternative long-read single molecule technologies (lrWGS) offer significant advantages for genome assembly and SV detection, while these technologies are currently cost prohibitive for large-scale disease studies and clinical diagnostics (∼5-12X higher cost than comparable coverage srWGS). Moreover, only dozens of such genomes are currently publicly accessible by comparison to millions of srWGS genomes that have been commissioned for international initiatives. Given this ubiquitous reliance on srWGS in human genetics and genomics, we sought to characterize and quantify the properties of SVs accessible to both srWGS and lrWGS to establish benchmarks and expectations in ongoing medical and population genetic studies, and to project the added value of SVs uniquely accessible to each technology. In analyses of three trios with matched srWGS and lrWGS from the Human Genome Structural Variation Consortium (HGSVC), srWGS captured ∼11,000 SVs per genome using reference-based algorithms, while haplotype-resolved assembly from lrWGS identified ∼25,000 SVs per genome. Detection power and precision for SV discovery varied dramatically by genomic context and variant class: 9.7% of the current GRCh38 reference is defined by segmental duplications (SD) and simple repeats (SR), yet 91.4% of deletions that were specifically discovered by lrWGS localized to these regions. Across the remaining 90.3% of the human reference, we observed extremely high concordance (93.8%) for deletions discovered by srWGS and lrWGS after error correction using the raw lrWGS reads. Conversely, lrWGS was superior for detection of insertions across all genomic contexts. Given that the non-SD/SR sequences span 90.3% of the GRCh38 reference, and encompass 95.9% of coding exons in currently annotated disease associated genes, improved sensitivity from lrWGS to discover novel and interpretable pathogenic deletions not already accessible to srWGS is likely to be incremental. However, these analyses highlight the added value of assembly-based lrWGS to create new catalogues of functional insertions and transposable elements, as well as disease associated repeat expansions in genomic regions previously recalcitrant to routine assessment.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans., Circulating lipoprotein(a) is an important risk factor for cardiovascular disease and shows variability between different ethnic groups. Here, Zekavat et al. perform whole-genome sequencing in individuals of European and African ancestries and find ancestry-specific genetic determinants for lipoprotein(a) levels.

Glioma is a unique neoplastic disease that develops exclusively in the central nervous system (CNS) and rarely metastasizes to other tissues. This feature strongly implicates the tumor-host CNS microenvironment in gliomagenesis and tumor progression. We investigated the differences and similarities in glioma biology as conveyed by transcriptomic patterns across four mammalian hosts: rats, mice, dogs, and humans. Given the inherent intra-tumoral molecular heterogeneity of human glioma, we focused this study on tumors with upregulation of the platelet-derived growth factor signaling axis, a common and early alteration in human gliomagenesis. The results reveal core neoplastic alterations in mammalian glioma, as well as unique contributions of the tumor host to neoplastic processes. Notable differences were observed in gene expression patterns as well as related biological pathways and cell populations known to mediate key elements of glioma biology, including angiogenesis, immune evasion, and brain invasion. These data provide new insights regarding mammalian models of human glioma, and how these insights and models relate to our current understanding of the human disease. NIH [T32 CA154274, F30-CA200345-01, R01CA139099, K12 NS080223, K08 NS090430]; Wallace Coulter Foundation; Passano Foundation; American Cancer Society-Research Scholar Grant [128970-RSG-16-012-01-CDD] This study was supported by NIH T32 CA154274 (NC), NIH F30-CA200345-01 (MBS), NIH R01CA139099 (JHR); the Wallace Coulter Foundation (JHR); the Passano Foundation (GW); NIH K12 NS080223 Neurosurgeon Research Career Development Program (GW), NIH K08 NS090430 (GW), American Cancer Society-Research Scholar Grant 128970-RSG-16-012-01-CDD (GW).

The prevailing genome assembly paradigm is to produce consensus sequences that “collapse” parental haplotypes into a consensus sequence. Here, we leverage the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing (Strand-seq)1,2 and combine them with high-fidelity (HiFi) long sequencing reads3, in a novel reference-free workflow for diploid de novo genome assembly. Employing this strategy, we produce completely phased de novo genome assemblies separately for each haplotype of a single individual of Puerto Rican origin (HG00733) in the absence of parental data. The assemblies are accurate (QV > 40), highly contiguous (contig N50 > 25 Mbp) with low switch error rates (0.4%) providing fully phased single-nucleotide variants (SNVs), indels, and structural variants (SVs). A comparison of Oxford Nanopore and PacBio phased assemblies identifies 150 regions that are preferential sites of contig breaks irrespective of sequencing technology or phasing algorithms.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (, Structural variants (SVs) in human genomes contribute diversity and diseases. Here, the authors use a multi-platform strategy to generate haplotype-resolved SVs for three human parent–child trios.

The clinical features of patients infected with pulmonary nontuberculous mycobacteria (PNTM) are well described, but the genetic components of infection susceptibility are not.To examine genetic variants in patients with PNTM, their unaffected family members, and a control group.Whole-exome sequencing was done on 69 white patients with PNTM and 18 of their white unaffected family members. We performed a candidate gene analysis using immune, cystic fibrosis transmembrance conductance regulator (CFTR), cilia, and connective tissue gene sets. The numbers of patients, family members, and control subjects with variants in each category were compared, as was the average number of variants per person.A significantly higher number of patients with PNTM than the other subjects had low-frequency, protein-affecting variants in immune, CFTR, cilia, and connective tissue categories (35, 26, 90, and 90%, respectively). Patients with PNTM also had significantly more cilia and connective tissue variants per person than did control subjects (2.47 and 2.55 compared with 1.38 and 1.40, respectively; P = 1.4 × 10(-6) and P = 2.7 × 10(-8), respectively). Patients with PNTM had an average of 5.26 variants across all categories (1.98 in control subjects; P = 2.8 × 10(-17)), and they were more likely than control subjects to have variants in multiple categories. We observed similar results for family members without PNTM infection, with the exception of the immune category.Patients with PNTM have more low-frequency, protein-affecting variants in immune, CFTR, cilia, and connective tissue genes than their unaffected family members and control subjects. We propose that PNTM infection is a multigenic disease in which combinations of variants across gene categories, plus environmental exposures, increase susceptibility to the infection.

The developing mammalian brain is destined for a female phenotype unless exposed to gonadal hormones during a perinatal sensitive period. It has been assumed that the undifferentiated brain is masculinized by direct induction of transcription by ligand-activated nuclear steroid receptors. We found that a primary effect of gonadal steroids in the highly sexually dimorphic preoptic area (POA) is to reduce activity of DNA methyltransferase (Dnmt) enzymes, thereby decreasing DNA methylation and releasing masculinizing genes from epigenetic repression. Pharmacological inhibition of Dnmts mimicked gonadal steroids, resulting in masculinized neuronal markers and male sexual behavior in female rats. Conditional knockout of the de novo Dnmt isoform, Dnmt3a, also masculinized sexual behavior in female mice. RNA sequencing revealed gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females. Our data show that brain feminization is maintained by the active suppression of masculinization via DNA methylation.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, and strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three human parent–child trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (

The human LINE-1 (or L1) element is a non-LTR retrotransposon that is mobilized through an RNA intermediate by an L1-encoded reverse transcriptase and other L1-encoded proteins. L1 elements remain actively mobile today and continue to mutagenize human genomes. Importantly, when new insertions disrupt gene function, they can cause diseases. Historically, L1s were thought to be active in the germline but silenced in adult somatic tissues. However, recent studies now show that L1 is active in at least some somatic tissues, including epithelial cancers. In this review, we provide an overview of these recent developments, and examine evidence that somatic L1 retrotransposition can initiate and drive tumorigenesis in humans. Recent studies have: (i) cataloged somatic L1 activity in many epithelial tumor types; (ii) identified specific full-length L1 source elements that give rise to somatic L1 insertions; and (iii) determined that L1 promoter hypomethylation likely plays an early role in the derepression of L1s in somatic tissues. A central challenge moving forward is to determine the extent to which L1 driver mutations can promote tumor initiation, evolution, and metastasis in humans.

In this review, we focus on progress that has been made with detecting small insertions and deletions (INDELs) in human genomes. Over the past decade, several million small INDELs have been discovered in human populations and personal genomes. The amount of genetic variation that is caused by these small INDELs is substantial. The number of INDELs in human genomes is second only to the number of single nucleotide polymorphisms (SNPs), and, in terms of base pairs of variation, INDELs cause similar levels of variation as SNPs. Many of these INDELs map to functionally important sites within human genes, and thus, are likely to influence human traits and diseases. Therefore, small INDEL variation will play a prominent role in personalized medicine.

SummaryTwo abundant classes of mobile elements, namely Alu and L1 elements, continue to generate new retrotransposon insertions in human genomes. Estimates suggest that these elements have generated millions of new germline insertions in individual human genomes worldwide. Unfortunately, current technologies are not capable of detecting most of these young insertions, and the true extent of germline mutagenesis by endogenous human retrotransposons has been difficult to examine. Here, we describe technologies for detecting these young retrotransposon insertions and demonstrate that such insertions indeed are abundant in human populations. We also found that new somatic L1 insertions occur at high frequencies in human lung cancer genomes. Genome-wide analysis suggests that altered DNA methylation may be responsible for the high levels of L1 mobilization observed in these tumors. Our data indicate that transposon-mediated mutagenesis is extensive in human genomes and is likely to have a major impact on human biology and diseases.

Proteins destined for import into the nucleus contain nuclear localization signals (NLSs) that are recognized by import receptors termed karyopherins or importins. Until recently, the only nuclear import sequence that had been well defined and characterized was the classical NLS (cNLS), which is recognized by importin α. However, Chook and coworkers (Lee, B. J., Cansizoglu, A. E., Süel, K. E., Louis, T. H., Zhang, Z., and Chook, Y. M. (2006) Cell 126, 543–558) have provided new insight into nuclear targeting with their identification of a novel NLS, termed the PY-NLS, that is recognized by the human karyopherin β2/transportin (Kapβ2) receptor. Here, we demonstrate that the PY-NLS is conserved in Saccharomyces cerevisiae and show for the first time that the PY-NLS is a functional nuclear targeting sequence in vivo. The apparent ortholog of Kapβ2 in yeast, Kap104, has two known cargos, the mRNA-binding proteins Hrp1 and Nab2, which both contain putative PY-NLS-like sequences. We find that the PY-NLS-like sequence within Hrp1, which closely matches the PY-NLS consensus, is both necessary and sufficient for nuclear import and is also required for receptor binding and protein function. In contrast, the PY-NLS-like sequences in Nab2, which vary from the PY-NLS consensus, are not required for proper import or protein function, suggesting that Kap104 may interact with different cargos using multiple mechanisms. Dissection of the PY-NLS consensus reveals that the minimal PY-NLS in yeast consists of the C-terminal portion of the human consensus, R/H/KX2–5PY, with upstream basic or hydrophobic residues enhancing the targeting function. Finally, we apply this analysis to a bioinformatic search of the yeast proteome as a preliminary search for new potential Kap104 cargos.

The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the beta-karyopherin importin beta, which mediates interactions with the nuclear pore complex, and the adaptor protein importin alpha, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.

Although human LINE-1 (L1) elements are actively mobilized in many cancers, a role for somatic L1 retrotransposition in tumor initiation has not been conclusively demonstrated. Here, we identify a novel somatic L1 insertion in the APC tumor suppressor gene that provided us with a unique opportunity to determine whether such insertions can actually initiate colorectal cancer (CRC), and if so, how this might occur. Our data support a model whereby a hot L1 source element on Chromosome 17 of the patient's genome evaded somatic repression in normal colon tissues and thereby initiated CRC by mutating the APC gene. This insertion worked together with a point mutation in the second APC allele to initiate tumorigenesis through the classic two-hit CRC pathway. We also show that L1 source profiles vary considerably depending on the ancestry of an individual, and that population-specific hot L1 elements represent a novel form of cancer risk.

Transposable genetic elements are abundant in the genomes of most organisms, including humans. These endogenous mutagens can alter genes, promote genomic rearrangements, and may help to drive the speciation of organisms. In this study, we identified almost 11,000 transposon copies that are differentially present in the human and chimpanzee genomes. Most of these transposon copies were mobilized after the existence of a common ancestor of humans and chimpanzees, approximately 6 million years ago. Alu, L1, and SVA insertions accounted for95% of the insertions in both species. Our data indicate that humans have supported higher levels of transposition than have chimpanzees during the past several million years and have amplified different transposon subfamilies. In both species, approximately 34% of the insertions were located within known genes. These insertions represent a form of species-specific genetic variation that may have contributed to the differential evolution of humans and chimpanzees. In addition to providing an initial overview of recently mobilized elements, our collections will be useful for assessing the impact of these insertions on their hosts and for studying the transposition mechanisms of these elements.

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.

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)

Nat. Neurosci. 18, 690–697 (2015); published online 30 March 2015; corrected after print 8 February 2017 In the version of this article initially published, the Whole Genome Bisulfite Sequencing (WGBS) analysis was based on a data set in which one of the female .bam alignment files (FV2) was mistakenly truncated.

Repetitive DNA is a significant component of eukaryotic genomes. We have developed a strategy to efficiently and accurately sequence repetitive DNA in the nematode Caenorhabditis elegans using integrated artificial transposons and automated fluorescent sequencing. Mapping and assembly tools represent important components of this strategy and facilitate sequence assembly in complex regions. We have applied the strategy to several cosmid assembly gaps resulting from repetitive DNA and have accurately recovered the sequences of these regions. Analysis of these regions revealed six novel transposon-like repetitive elements, IR-1, IR-2, IR-3, IR-4, IR-5, and TR-1. Each of these elements represents a middle-repetitive DNA family in C. elegans containing at least 3–140 copies per genome. Copies of IR-1, IR-2, IR-4, and IR-5 are located on all (or most) of the six nematode chromosomes, whereas IR-3 is predominantly located on chromosome X. These elements are almost exclusively interspersed between predicted genes or within the predicted introns of these genes, with the exception of a single IR-5 element, which is located within a predicted exon. IR-1, IR-2, and IR-3 are flanked by short sequence duplications resembling the target site duplications of transposons. We have established a website database (http://www.welch.jhu.edu/~devine/RepDNAdb.html) to track and cross-reference these transposon-like repetitive elements that contains detailed information on individual element copies and provides links to appropriate GenBank records. This set of tools may be used to sequence, track, and study repetitive DNA in model organisms and humans.[The sequences reported in this paper have been deposited in GenBank under accession nos. U53139 and U86946–U86951.]

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.

Genomic silencing is a fundamental mechanism of transcriptional regulation, yet little is known about conserved mechanisms of silencing. We report here the discovery of four Saccharomyces cerevisiae homologs of the SIR2 silencing gene (HSTs), as well as conservation of this gene family from bacteria to mammals. At least three HST genes can function in silencing; HST1 overexpression restores transcriptional silencing to a sir2 mutant and hst3 hst4 double mutants are defective in telomeric silencing. In addition, HST3 and HST4 together contribute to proper cell cycle progression, radiation resistance, and genomic stability, establishing new connections between silencing and these fundamental cellular processes.

Mammalian cells displaying the multidrug resistance (mdr) phenotype are refractory to the toxic effects of a group of unrelated natural product drugs, many of which are used for cancer chemotherapy. The pattern of cross-resistance can be extremely variable among independently selected cell lines, even though such cells are often exposed to only a single drug. The overexpression of P-glycoprotein (pgp), a 150-180-kDa drug efflux pump, has been shown to confer mdr to otherwise drug-sensitive cells; however, the variable nature of cross-resistance indicates that normal pgps alone are unlikely to account for all of the observed cross-resistance phenotypes. In this report, we examined possible factors contributing to cross-resistance diversity in mammalian cells. We show that drug-resistant Chinese hamster lung cells selected during relatively short periods of drug exposure in vitro (less than 6-8 weeks) routinely overexpressed endogenous pgps and predominantly showed a cross-resistance pattern that was similar to that conferred by the introduction and overexpression of the hamster wild-type pgp1 cDNA alone. Longer drug exposure periods at higher drug concentrations, however, led to the selection of cell lines with altered cross-resistance properties. Like the short term clones, these cell lines all overexpressed endogenous pgp. In one case, the altered phenotype was shown to be caused by the acquisition of point mutations within codons 338 and 339 of the pgp1 gene, leading to two adjacent amino acid substitutions within the encoded pgp. Although the basis for the remaining altered phenotypes remains unknown, these results indicate that additional genetic alterations beyond those responsible for the initial acquisition of mdr emerge in the face of increased selective pressure, thus further modifying or complementing the cross-resistance phenotype initially conferred by wild-type pgp.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.

Human genetic variation is expected to play a central role in personalized medicine. Yet only a fraction of the natural genetic variation that is harbored by humans has been discovered to date. Here we report almost 2 million small insertions and deletions (INDELs) that range from 1 bp to 10,000 bp in length in the genomes of 79 diverse humans. These variants include 819,363 small INDELs that map to human genes. Small INDELs frequently were found in the coding exons of these genes, and several lines of evidence indicate that such variation is a major determinant of human biological diversity. Microarray-based genotyping experiments revealed several interesting observations regarding the population genetics of small INDEL variation. For example, we found that many of our INDELs had high levels of linkage disequilibrium (LD) with both HapMap SNPs and with high-scoring SNPs from genome-wide association studies. Overall, our study indicates that small INDEL variation is likely to be a key factor underlying inherited traits and diseases in humans.

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best-characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin-α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9-12 amino acid linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 amino acid linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 amino acid linker. Our studies reveal that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition

In an effort to better understand the preferential resistance to actinomycin D displayed by the multidrug-resistant Chinese hamster lung cell line DC-3F/ADX, we have cloned from those cells a number of cDNAs representing p-glycoprotein gene transcripts. Of the 12 clones isolated, all represent pgp1 transcripts and one, pADX165, contains a 4304-base pair insert with an open reading frame encoding a 1276-amino acid protein that is the homolog of the mouse mdr3/mdr1a gene product. A domain by domain comparison of this protein with p-glycoproteins capable of supporting multidrug resistance, i.e. human mdr1, mouse mdr1/mdr1b, and mouse mdr3/mdr1a, shows that, in addition to the ATP binding sites, the second, fourth, and eleventh transmembrane domains and the four small intracellular loops, IC-1, IC-2, IC-4, and IC-5, are highly conserved and are therefore likely to be important for the maintenance of p-glycoprotein function. Of the remaining 11 cDNA clones, 9 were found to be truncated versions of pADX165. Two others, however, pADX185 and pADX124, contained internal deletions resulting in open reading frames capable of encoding lnovel forms of p-glycoprotein. S1 nuclease and RNase protection analysis demonstrated that these cDNAs represent transcripts present in a number of different multidrug-resistant Chinese hamster lung cell lines. Hence, both are considered to be splicing variants of the hamster pgp1 gene primary transcript.

Like its retroviral relatives, the long terminal repeat retrotransposon Ty1 in the yeast Saccharomyces cerevisiae must traverse a permanently intact nuclear membrane for successful transposition and replication. For retrotransposition to occur, at least a subset of Ty1 proteins, including the Ty1 integrase, must enter the nucleus. Nuclear localization of integrase is dependent upon a C-terminal nuclear targeting sequence. However, the nuclear import machinery that recognizes this nuclear targeting signal has not been defined. We investigated the mechanism by which Ty1 integrase gains access to nuclear DNA as a model for how other retroelements, including retroviruses like HIV, may utilize cellular nuclear transport machinery to import their essential nuclear proteins. We show that Ty1 retrotransposition is significantly impaired in yeast mutants that alter the classical nuclear protein import pathway, including the Ran-GTPase, and the dimeric import receptor, importin-alpha/beta. Although Ty1 proteins are made and processed in these mutant cells, our studies reveal that an integrase reporter is not properly targeted to the nucleus in cells carrying mutations in the classical nuclear import machinery. Furthermore, we demonstrate that integrase coimmunoprecipitates with the importin-alpha transport receptor and directly binds to importin-alpha. Taken together, these data suggest Ty1 integrase can employ the classical nuclear protein transport machinery to enter the nucleus.

Although many studies have been conducted to identify single nucleotide polymorphisms (SNPs) in humans, few studies have been conducted to identify alternative forms of natural genetic variation, such as insertion and deletion (INDEL) polymorphisms. In this report, we describe an initial map of human INDEL variation that contains 415,436 unique INDEL polymorphisms. These INDELs were identified with a computational approach using DNA re-sequencing traces that originally were generated for SNP discovery projects. They range from 1 bp to 9989 bp in length and are split almost equally between insertions and deletions, relative to the chimpanzee genome sequence. Five major classes of INDELs were identified, including (1) insertions and deletions of single-base pairs, (2) monomeric base pair expansions, (3) multi-base pair expansions of 2–15 bp repeat units, (4) transposon insertions, and (5) INDELs containing random DNA sequences. Our INDELs are distributed throughout the human genome with an average density of one INDEL per 7.2 kb of DNA. Variation hotspots were identified with up to 48-fold regional increases in INDEL and/or SNP variation compared with the chromosomal averages for the same chromosomes. Over 148,000 INDELs (35.7%) were identified within known genes, and 5542 of these INDELs were located in the promoters and exons of genes, where gene function would be expected to be influenced the greatest. All INDELs in this study have been deposited into dbSNP and have been integrated into maps of human genetic variation that are available to the research community.

Although a large proportion (44%) of the human genome is occupied by transposons and transposon-like repetitive elements, only a small proportion (0.05%) of these elements remain active today. Recent evidence indicates that approximately 35-40 subfamilies of Alu, L1 and SVA elements (and possibly HERV-K elements) remain actively mobile in the human genome. These active transposons are of great interest because they continue to produce genetic diversity in human populations and also cause human diseases by integrating into genes. In this review, we examine these active human transposons and explore mechanistic factors that influence their mobilization.

Transposons and transposon-like repetitive elements collectively occupy 44% of the human genome sequence. In an effort to measure the levels of genetic variation that are caused by human transposons, we have developed a new method to broadly detect transposon insertion polymorphisms of all kinds in humans. We began by identifying 606,093 insertion and deletion (indel) polymorphisms in the genomes of diverse humans. We then screened these polymorphisms to detect indels that were caused by de novo transposon insertions. Our method was highly efficient and led to the identification of 605 nonredundant transposon insertion polymorphisms in 36 diverse humans. We estimate that this represents 25–35% of ∼2075 common transposon polymorphisms in human populations. Because we identified all transposon insertion polymorphisms with a single method, we could evaluate the relative levels of variation that were caused by each transposon class. The average human in our study was estimated to harbor 1283 Alu insertion polymorphisms, 180 L1 polymorphisms, 56 SVA polymorphisms, and 17 polymorphisms related to other forms of mobilized DNA. Overall, our study provides significant steps toward (i) measuring the genetic variation that is caused by transposon insertions in humans and (ii) identifying the transposon copies that produce this variation.

An international effort is underway to generate a comprehensive haplotype map (HapMap) of the human genome represented by an estimated 300,000 to 1 million 'tag' single nucleotide polymorphisms (SNPs). Our analysis indicates that the current human SNP map is not sufficiently dense to support the HapMap project. For example, 24.6% of the genome currently lacks SNPs at the minimal density and spacing that would be required to construct even a conservative tag SNP map containing 300,000 SNPs. In an effort to improve the human SNP map, we identified 140,696 additional SNP candidates using a new bioinformatics pipeline. Over 51,000 of these SNPs mapped to the largest gaps in the human SNP map, leading to significant improvements in these regions. Our SNPs will be immediately useful for the HapMap project, and will allow for the inclusion of many additional genomic intervals in the final HapMap. Nevertheless, our results also indicate that additional SNP discovery projects will be required both to define the haplotype architecture of the human genome and to construct comprehensive tag SNP maps that will be useful for genetic linkage studies in humans.

Retroviruses and their relatives, the long terminal repeat (LTR) retrotransposons, carry out complex life cycles within the cells of their hosts. We have exploited a collection of gene deletion mutants developed by the Saccharomyces Genome Deletion Project to perform a functional genomics screen for host factors that influence the retrovirus-like Ty1 element in yeast. A total of 101 genes that presumably influence many different aspects of the Ty1 retrotransposition cycle were identified from our analysis of 4483 homozygous diploid deletion strains. Of the 101 identified mutants, 46 had significantly altered levels of Ty1 cDNA, whereas the remaining 55 mutants had normal levels of Ty1 cDNA. Thus, approximately half of the mutants apparently affected the early stages of retrotransposition leading up to the assembly of virus-like particles and cDNA replication, whereas the remaining half affected steps that occur after cDNA replication. Although most of the mutants retained the ability to target Ty1 integration to tRNA genes, 2 mutants had reduced levels of tRNA gene targeting. Over 25% of the gene products identified in this study were conserved in other organisms, suggesting that this collection of host factors can serve as a starting point for identifying host factors that influence LTR retroelements and retroviruses in other organisms. Overall, our data indicate that Ty1 requires a large number of cellular host factors to complete its retrotransposition cycle efficiently.

Retrotransposon Ty1 faces a formidable cell barrier during transposition--the yeast nuclear membrane which remains intact throughout the cell cycle. We investigated the mechanism by which transposition intermediates are transported from the cytoplasm (the presumed site of Ty1 DNA synthesis) to the nucleus, where they are integrated into the genome. Ty1 integrase has a nuclear localization signal (NLS) at its C terminus. Both full-length integrase and a C-terminal fragment localize to the nucleus. C-terminal deletion mutants in Ty1 integrase were used to map the putative NLS to the last 74 amino acid residues of integrase. Mutations in basic segments within this region decreased retrotransposition at least 50-fold in vivo. Furthermore, these mutant integrase proteins failed to localize to the nucleus. Production of virus-like particles, reverse transcriptase activity, and complete in vitro Ty1 integration resembled wild-type levels, consistent with failure of the mutant integrases to enter the nucleus.

Retroviruses and their relatives, the LTR-containing retrotransposons, integrate newly replicated cDNA copies of their genomes into the genomes of their hosts using element-encoded integrases. Although target site selection is not well understood for this general class of elements, it is becoming clear that some elements target their integration events to very specific regions of their host genomes. Evidence is accumulating that the yeast retrotransposon Ty1 behaves in this manner. Ty1 is found frequently adjacent to tRNA genes in the yeast genome and experimental evidence implicates these regions as preferred integration sites. To determine the basis for Ty1 targeting, we developed an in vivo integration assay using a Ty1 donor plasmid and a second target plasmid that could be used to measure the relative frequency of Ty1 integration into sequences cloned from various regions of the yeast genome. Targets containing genes transcribed by RNA polymerase III (Pol III) were up to several hundredfold more active as integration targets than "cold" sequences lacking such genes. High-frequency targeting was dependent on Pol III transcription, and integration was "region specific," occurring exclusively upstream of the transcription start sites of these genes. Thus, Ty1 has evolved a powerful targeting mechanism, requiring Pol III transcription to integrate its DNA at very specific locations within the yeast genome.

Hamster cells grown in culture may, like human and mouse cells, develop multidrug resistance (MDR) when exposed to certain cytotoxic chemotherapeutic agents. Several phenotypic features that are characteristic of MDR have been described; these include (1) resistance to many structurally and functionally unrelated drugs that have different cellular targets and modes of action; (2) reversal of MDR by certain agents, including verapamil and cyclosporin A; and (3) reduced intracellular drug accumulation relative to that of drug-sensitive cells. In this report we show that the introduction and overexpression of the hamster pgp1 cDNA confers to otherwise drug-sensitive cells an MDR phenotype with these features. Moreover, pgp1 transfectants showed varying degrees of resistance to anthracycline analogues, indicating that structural analogues of commonly used anticancer agents are capable of circumventing drug resistance conferred by pgp.

Eukaryotic cells can display resistance to a wide range of natural-product chemotheraputic agents by the expression of P-glycoprotein (pgp), a putative plasma membrane transporter that is thought to mediate the efflux of these agents from cells. We have identified, in cells selected for multidrug resistance with actinomycin D, a mutant form of pgp that contains two amino acid substitutions within the putative sixth transmembrane domain. In transfection experiments, this altered pgp confers a cross-resistance phenotype that is altered significantly from that conferred by the normal protein, displaying maximal resistance to actinomycin D. These results strongly implicate the sixth transmembrane domain in the mechanism of pgp drug recognition and efflux. Moreover, they indicate a close functional homology between pgp and the cystic fibrosis transmembrane regulator in which the sixth transmembrane domain has also been shown to influence substrate specificity.