Ryan E. Mills

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 Structural Variation Analysis Group of The 1000 Genomes Project reports an integrated structural variation map based on discovery and genotyping of eight major structural variation classes in 2,504 unrelated individuals from across 26 populations; structural variation is compared within and between populations and its functional impact is quantified. The Structural Variation Analysis Group of The 1000 Genomes Project reports an integrated structural variation map based on discovery and genotyping of eight major structural variation classes in genomes for 2,504 unrelated individuals from across 26 populations. They characterize structural variation within and between populations and quantify its functional effect. The authors further create a phased reference panel that will be valuable for population genetic and disease association studies.

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 β-karyopherin importin β, which mediates interactions with the nuclear pore complex, and the adaptor protein importin α, 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. 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 β-karyopherin importin β, which mediates interactions with the nuclear pore complex, and the adaptor protein importin α, 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. In eukaryotic cells, the genetic material and transcriptional machinery of the nucleus are separated from the translational machinery and metabolic systems of the cytoplasm by the nuclear envelope. This segregation facilitates the precise regulation of cellular processes such as gene expression (1Kaffman A. O'Shea E.K. Annu. Rev. Cell Dev. Biol. 1999; 15: 291-339Crossref PubMed Scopus (261) Google Scholar), signal transduction (2Johnson H.M. Subramaniam P.S. Olsnes S. Jans D.A. Bioessays. 2004; 26: 993-1004Crossref PubMed Scopus (92) Google Scholar), and cell cycle progression (3Cyert M.S. J. Biol. Chem. 2001; 276: 20805-20808Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) through selective regulation of bidirectional transport between the nucleus and the cytoplasm. However, this physical separation also necessitates the existence of molecular machinery that specifically recognizes cargo in one compartment, translocates it through the nuclear pore, and releases it in the other compartment. Nuclear transport systems of this kind were first proposed when a nuclear targeting signal in the simian virus 40 (SV40) 2The abbreviations used are: SV40, simian virus 40; NLS, nuclear localization signal; cNLS, classical nuclear localization signal; IBB, importin β-binding; GFP, green fluorescent protein. large T antigen was characterized more than 20 years ago (4Kalderon D. Richardson W.D. Markham A.F. Smith A.E. Nature. 1984; 311: 33-38Crossref PubMed Scopus (910) Google Scholar, 5Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1874) Google Scholar). Since then, several pathways for nucleocytoplasmic transport have been described, of which the classical nuclear import pathway is the best characterized. The integration of detailed structural information on the components of the pathway, whole genome surveys, and extensive molecular analysis has generated powerful insight into the crucial interactions that underlie this pathway. Here we review recent studies that have defined key aspects of the cargo/import receptor interaction in the classical nuclear import cycle and present results of a bioinformatics-based assessment of the likely prevalence of this system within the model eukaryotic organism, Saccharomyces cerevisiae. Transport of macromolecules into and out of the nucleus occurs through large, proteinaceous structures called nuclear pore complexes (NPCs) (6Fahrenkrog B. Aebi U. Nat. Rev. Mol. Cell. Biol. 2003; 4: 757-766Crossref PubMed Scopus (336) Google Scholar, 7Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (299) Google Scholar, 8Allen T.D. Cronshaw J.M. Bagley S. Kiseleva E. Goldberg M.W. J. Cell Sci. 2000; 113: 1651-1659Crossref PubMed Google Scholar, 9Fahrenkrog B. Aebi U. Results Probl. Cell Differ. 2002; 35: 25-48Crossref PubMed Scopus (17) Google Scholar). Nuclear pore complexes allow passive diffusion of ions and small proteins (<40 kDa) but restrict passage of larger molecules to those containing an appropriate targeting signal (10Paine P.L. Moore L.C. Horowitz S.B. Nature. 1975; 254: 109-114Crossref PubMed Scopus (583) Google Scholar, 11Bonner W.M. Protein Migration and Accumulation in Nuclei, Academic Press, New York. 1978; Google Scholar). The pores are constructed from a class of proteins called “nucleoporins,” a subset of which contains a tandem series of phenylalanine-glycine (FG) repeats that line the central transport channel of the pore (8Allen T.D. Cronshaw J.M. Bagley S. Kiseleva E. Goldberg M.W. J. Cell Sci. 2000; 113: 1651-1659Crossref PubMed Google Scholar, 12Stewart M. Baker R.P. Bayliss R. Clayton L. Grant R.P. Littlewood T. Matsuura Y. 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Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar), respectively. This distinction may be artificial though, because in an ideal system, a transport factor would import one molecule, switch to export mode, and return to the cytoplasm carrying a different cargo. For example, both import and export cargos for the S. cerevisiae Msn5 receptor have been identified (18Yoshida K. Blobel G. J. Cell Biol. 2001; 152: 729-740Crossref PubMed Scopus (109) Google Scholar, 19Kaffman A. Rank N.M. O'Neill E.M. Huang L.S. O'Shea E.K. Nature. 1998; 396: 482-486Crossref PubMed Scopus (288) Google Scholar, 20Blondel M. Alepuz P.M. Huang L.S. Shaham S. Ammerer G. Peter M. Genes Dev. 1999; 13: 2284-2300Crossref PubMed Scopus (81) Google Scholar, 21DeVit M.J. Johnston M. Curr. Biol. 1999; 9: 1231-1241Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 22Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Many transport receptors are members of the importin β superfamily and are called collectively “β-karyopherins.” Cargo proteins can bind directly to β-karyopherins; however, in classical nuclear import, the interaction between the β-karyopherin and the cargo is mediated by the adaptor molecule importin α. Recent modeling studies have shown that addition of an adaptor to the system results in lower transport efficiency, but this feature also may facilitate the formation of a higher nuclear cytoplasmic gradient (23Riddick G. Macara I.G. J. Cell Biol. 2005; 168: 1027-1038Crossref PubMed Scopus (98) Google Scholar). The energy for nuclear transport is provided by the small Ras family GTPase, Ran (24Quimby B.B. Dasso M. Curr. Opin. Cell Biol. 2003; 15: 338-344Crossref PubMed Scopus (162) Google Scholar). Like other GTPases (25Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1844) Google Scholar), Ran cycles between a GTP- and a GDP-bound state. The nucleotide state of Ran is modulated by regulatory proteins, primarily the Ran guanine nucleotide exchange factor (RanGEF) in the nucleus and the Ran GTPase-activating protein (RanGAP) in the cytoplasm (26Corbett A.H. Koepp D.M. Lee M.S. Schlenstedt G. Hopper A.K. Silver P.A. J. Cell Biol. 1995; 130: 1017-1026Crossref PubMed Scopus (152) Google Scholar, 27Becker J. Melchior F. Gerke V. Bischoff F.R. Ponstingl H. Wittinghofer A. J. Biol. Chem. 1995; 270: 11860-11865Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 28Klebe C. Prinz H. Wittinghofer A. Goody R.S. Biochemistry. 1995; 34: 12543-12552Crossref PubMed Scopus (194) Google Scholar, 29Bischoff F.R. Ponstingl H. Nature. 1991; 354: 80-82Crossref PubMed Scopus (541) Google Scholar). Because these key regulatory factors are compartmentalized, the different forms of Ran are asymmetrically distributed in the cell, with RanGTP enriched in the nucleus and RanGDP enriched in the cytoplasm (30Kalab P. Weis K. Heald R. Science. 2002; 295: 2452-2456Crossref PubMed Scopus (443) Google Scholar, 31Smith A.E. Slepchenko B.M. Schaff J.C. Loew L.M. Macara I.G. Science. 2002; 295: 488-491Crossref PubMed Scopus (162) Google Scholar). This compartmentalization allows Ran to impart directionality to nuclear transport by acting as a molecular switch that controls the binding and release of cargo. Therefore, import receptors bind cargo in the cytoplasm in the absence of RanGTP and release cargo in the nucleus upon RanGTP binding to the complex. In contrast, export receptors bind cargo in the nucleus in complex with RanGTP with hydrolysis to GDP in the cytoplasm triggering dissociation. The best understood pathway of nucleocytoplasmic transport is the classical nuclear import pathway (Fig. 1). Here, importin α recognizes and binds cargo in the cytoplasm, linking it to the β-karyopherin, importin β (32Goörlich D. Kostka S. Kraft R. Dingwall C. Laskey R.A. Hartmann E. Prehn S. Curr. Biol. 1995; 5: 383-392Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). Importin β then mediates interaction of the trimeric complex with the nuclear pore as it translocates into the nucleus. Once the import complex reaches the nucleus, it is dissociated by RanGTP. Binding of RanGTP to importin β causes a conformational change that results in the release of the importin α-cargo complex (33Lee S.J. Matsuura Y. Liu S.M. Stewart M. Nature. 2005; 435: 693-696Crossref PubMed Scopus (171) Google Scholar). An autoinhibitory region on the importin β-binding (IBB) domain of importin α (34Kobe B. Nat. Struct. Biol. 1999; 6: 301-304Crossref PubMed Scopus (19) Google Scholar, 35Harreman M.T. Hodel M.R. Fanara P. Hodel A.E. Corbett A.H. J. Biol. Chem. 2003; 278: 5854-5863Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), the nucleoporin Nup2 (Nup50 or Npap60 in vertebrates) (36Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 37Matsuura Y. Lange A. Harreman M.T. Corbett A.H. Stewart M. EMBO J. 2003; 22: 5358-5369Crossref PubMed Scopus (84) Google Scholar, 38Matsuura Y. Stewart M. EMBO J. 2005; 24: 3681-3689Crossref PubMed Scopus (121) Google Scholar), and the export receptor for importin α, Cse1/RanGTP (CAS/RanGTP in vertebrates) (39Matsuura Y. Stewart M. Nature. 2004; 432: 872-877Crossref PubMed Scopus (164) Google Scholar) then work together to deliver the cargo into the nucleus. Finally, Cse1/RanGTP recycles importin α back to the cytoplasm in preparation for another round of import (40Hood J.K. Silver P.A. J. Biol. Chem. 1998; 273: 35142-35146Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 41Kutay U. Bischoff F.R. Kostka S. Kraft R. Goörlich D. Cell. 1997; 90: 1061-1071Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). The first step of nuclear import occurs when an importin discriminates between its cargo and other cellular proteins. Proteins destined for transport into the nucleus contain amino acid targeting sequences called nuclear localization signals (NLSs). The best characterized transport signal is the classical NLS (cNLS) for nuclear protein import, which consists of either one (monopartite) or two (bipartite) stretches of basic amino acids (4Kalderon D. Richardson W.D. Markham A.F. Smith A.E. Nature. 1984; 311: 33-38Crossref PubMed Scopus (910) Google Scholar, 42Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1249) Google Scholar, 43Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1713) Google Scholar). Monopartite cNLSs are exemplified by the SV40 large T antigen NLS (126PKKKRRV132) and bipartite cNLSs are exemplified by the nucleoplasmin NLS (155KRPAATKKAGQAKKKK170). Consecutive residues from the N-terminal of the NLS are referred to as E. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M.R. T. B. J. Mol. Biol. 2000; PubMed Scopus Google Scholar) and M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) studies have identified of the key for a These studies that a a in the by basic residues in and to a of through of the SV40, and artificial bipartite SV40 it was that the binding of a for importin α in with the state nuclear and import of the cargo in M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, A.E. Harreman M.T. Hodel M.R. Corbett A.H. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). These for the binding of a cNLS, a has a binding of for importin α. sequences either bind importin α to be imported or to be from the receptor in the nucleus, release factors such as Nup2 and can in this cargo release step (36Gilchrist D. Mykytka B. Rexach M. J. Biol. Chem. 2002; 277: 18161-18172Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 37Matsuura Y. Lange A. Harreman M.T. Corbett A.H. Stewart M. EMBO J. 2003; 22: 5358-5369Crossref PubMed Scopus (84) Google Scholar, 38Matsuura Y. Stewart M. EMBO J. 2005; 24: 3681-3689Crossref PubMed Scopus (121) Google Scholar, Y. Stewart M. Nature. 2004; 432: 872-877Crossref PubMed Scopus (164) Google Scholar, D. Rexach M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Recent also that the import of cargo on the of formation of the import complex (23Riddick G. Macara I.G. J. Cell Biol. 2005; 168: 1027-1038Crossref PubMed Scopus (98) Google Scholar). Therefore, the import and of cargo in the nucleus is by both the of the cargo for importin α and by the of the importin α receptor (23Riddick G. Macara I.G. J. Cell Biol. 2005; 168: 1027-1038Crossref PubMed Scopus (98) Google Scholar, A.E. Harreman M.T. Hodel M.R. Corbett A.H. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). This is also of other NLS interactions B.L. J. R. J. Cell Biol. 2006; PubMed Scopus Google Scholar). The molecular for of a by importin α has been defined E. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M.R. T. B. J. Mol. Biol. 2000; PubMed Scopus Google Scholar, E. M. L. Blobel G. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M.R. T. G. A. Jans D.A. B. Biochem. J. 2003; PubMed Scopus Google Scholar). These structural studies that importin α is of a large domain consisting of of which is constructed from (Fig. and a N-terminal domain for both binding to importin β (32Goörlich D. Kostka S. Kraft R. Dingwall C. Laskey R.A. Hartmann E. Prehn S. Curr. Biol. 1995; 5: 383-392Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar) and cargo (34Kobe B. Nat. Struct. Biol. 1999; 6: 301-304Crossref PubMed Scopus (19) Google Scholar, P. Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M.T. Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The of repeats a molecule the and are within a on the are by together with a of residues (34Kobe B. Nat. Struct. Biol. 1999; 6: 301-304Crossref PubMed Scopus (19) Google Scholar, E. M. L. Blobel G. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The which the binds cNLSs and the larger of basic residues in bipartite The is more and binds the of basic residues in bipartite bind to the in an with to the of the importin α The key then between the of the and with residues the binding the key (34Kobe B. Nat. Struct. Biol. 1999; 6: 301-304Crossref PubMed Scopus (19) Google Scholar, E. M. L. Blobel G. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This binding which is for importin α with of its cargos (34Kobe B. Nat. Struct. Biol. 1999; 6: 301-304Crossref PubMed Scopus (19) Google Scholar, E. M. L. Blobel G. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), allows an autoinhibitory region within importin α to specifically for binding with to the of cargo in the nucleus (Fig. This autoinhibitory consists of residues within the N-terminal domain of importin α M.T. Hodel M.R. Fanara P. Hodel A.E. Corbett A.H. J. Biol. Chem. 2003; 278: 5854-5863Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, M.T. Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) that the basic of the trimeric import complex into the nucleus, the N-terminal domain of importin α is by importin β, it to and with the on the of importin α (34Kobe B. Nat. Struct. Biol. 1999; 6: 301-304Crossref PubMed Scopus (19) Google Scholar, E. M. L. Blobel G. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M.R. T. G. A. Jans D.A. B. Biochem. J. 2003; PubMed Scopus Google Scholar). Because both and bipartite cNLSs bind to the the autoinhibitory region the binding of both of cNLS, the release of cargo within the nucleus. The classical NLS is of as the NLS. was the first NLS to be and as of proteins the classical import pathway have been characterized. Because of the of proteins, have that this pathway is the in the however, studies have the of cargos imported this is that other pathways for a large of nuclear and that are because the for and of are this we have of the S. cerevisiae genome and sequences for and bipartite cNLSs to the prevalence of classical For this the from K. P. Trends Biochem. Sci. 1999; 24: Full Text Full Text PDF PubMed Scopus Google Scholar) for cNLSs and bipartite cNLSs was used to of we the by the proteins in the S. cerevisiae D.A. J. 2006; 34: PubMed Scopus Google Scholar) to the of proteins predicted to contain a which have the to the nucleus the classical import pathway. we the proteins to either the nucleus or the in a localization that was with a Gerke L.C. O'Shea E.K. Nature. 2003; PubMed Scopus Google Scholar). that these proteins were to the nucleus by nuclear import pathway and that a used classical nuclear we the proteins that with importin α to the C. T. L. A. M. 2006; 34: PubMed Scopus Google Scholar), because proteins that contain cNLSs with importin α. that the bipartite consists of the a and basic proteins that contain a bipartite by contain a proteins containing a bipartite were from the The results of this analysis are in of the and are in In the of proteins, proteins contain a predicted bipartite cNLS, and contain a predicted This that classical nuclear import may be as as the because of the proteins in the cell have the to the nucleus the classical nuclear import pathway. This is a because of proteins to the nucleus state when with Gerke L.C. O'Shea E.K. Nature. 2003; PubMed Scopus Google Scholar). the proteins that have been to the nucleus in the contain a bipartite and contain a Therefore, of nuclear proteins are predicted to classical nuclear import, may other to the nucleus. In the of proteins that with importin α, contain a predicted bipartite and contain a predicted cNLS, that contain predicted this because cNLSs are to interactions with importin however, of the interactions in the are genetic in and or a physical In proteins that with importin α in are such as importin β import for importin and export factor for importin which with importin α through (39Matsuura Y. Stewart M. Nature. 2004; 432: 872-877Crossref PubMed Scopus (164) Google Scholar, G. C. Weis K. Nature. 1999; PubMed Scopus Google Scholar). The of a also may because of these proteins may contain cNLSs and importin import but by the For example, the of the and of protein contain a classical targeting however, upon basic residues that a by importin α R. K. L. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). a large of the proteins in these for import by the classical system, our analysis collectively that nuclear localization also are likely to be studies and be to these These and bioinformatics are for because can pathways of import and a for how a protein of interest the nucleus. a into the of nuclear transport by the of in the import of protein. However, can residues that may a a can be a targeting it M. Silver P.A. Corbett A.H. 2002; PubMed Scopus Google Scholar). the be for import, that transport of the protein of interest into the nucleus is when the is The approach to a is is to one or more of the residues to and the nuclear localization of the protein the be to an protein to the nucleus. the is to the of that the of the in the protein of interest and the of the protein is the protein of interest directly with its import and this interaction be mediated by the identified The best to this is to in binding with proteins in the of either RanGDP or RanGTP. complexes are dissociated by in these binding the protein of interest with its import receptor in the of RanGDP but in the of RanGTP. to which import pathway the protein of interest in it be that the nuclear transport machinery in import of the protein. For example, in S. the localization of the protein of interest be the in a containing a of the gene either importin α or importin the import pathway is more in systems because specific of nuclear import have been a has these then it may be a NLS for the protein of However, it is to that a specific protein may have more than one nuclear targeting that may be used by different pathways or to different of the and of nuclear transport in vivo. In this we have the of classical NLS cargo in by the of a and the of how it with its import importin α. have also the prevalence of the classical nuclear import system in the cell by the of proteins that contain a predicted However, binding cargo is the first step in an complex and recent studies have on of the other of classical import such as the cargo release the of importin α with the release factor Nup2 has been Y. Lange A. Harreman M.T. Corbett A.H. Stewart M. EMBO J. 2003; 22: 5358-5369Crossref PubMed Scopus (84) Google Scholar, 38Matsuura Y. Stewart M. EMBO J. 2005; 24: 3681-3689Crossref PubMed Scopus (121) Google Scholar), and importin α the of importin α with its export receptor has been (39Matsuura Y. Stewart M. Nature. 2004; 432: 872-877Crossref PubMed Scopus (164) Google Scholar). studies on one of the of the of cargo through the nuclear a is understood the classical nuclear import pathway, is the of other transport Lee A.E. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) have to this and have proposed for NLS by as structural studies with bioinformatics to the one facilitate the of the of interaction between other and of a more understanding of nucleocytoplasmic transport in vivo.

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 (<50 bp) and 27,622 SVs (≥50 bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.

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.