Pericentromeric regions of human chromosomes are composed of tandem-repeated and highly organized sequences named satellite DNAs. Human classical satellite DNAs are classified into three families named HSat1, HSat2, and HSat3, which have historically posed a challenge for the assembly of the human reference genome where they are misrepresented due to their repetitive nature. Although being known for a long time as the most AT-rich fraction of the human genome, classical satellite HSat1A has been disregarded in genomic and transcriptional studies, falling behind other human satellites in terms of functional knowledge. Here, we aim to characterize and provide an understanding on the biological relevance of HSat1A.

Human recombination rates vary along the chromosomes as well as between the two sexes. There is growing evidence that epigenetic factors may have an important influence on recombination rates, as well as on crossover position. Using both public database analysis and wet-bench approaches, we revisited the relationship between increased rates of meiotic recombination and genome imprinting. We constructed metric linkage disequilibrium (LD) maps for all human chromosomal regions known to contain one or more imprinted genes. We show that imprinted regions contain significantly more LD units (LDU) and have significantly more haplotype blocks of smaller sizes than flanking nonimprinted regions. There is also an excess of hot-spots of recombination at imprinted regions, and this is likely to do with the presence of imprinted genes, per se. These findings indicate that imprinted chromosomal regions are historical "hot-spots" of recombination. We also demonstrate, by direct segregation analysis at the 11p15.5 imprinted region, that there is remarkable agreement between sites of meiotic recombination and steps in LD maps. Although the increase in LDU/Megabase at imprinted regions is not associated with any significant enrichment for any particular sequence class, major sequence determinants of recombination rates seem to differ between imprinted and control regions. Interestingly, fine-mapping of recombination events within the most male meiosis-specific recombination hot-spot of Chromosome 11p15.5 indicates that many events may occur within or directly adjacent to regions that are differentially methylated in somatic cells. Taken together, these findings support the involvement of a combination of specific DNA sequences and epigenetic factors as major determinants of hot-spots of recombination at imprinted chromosomal regions.

Presented here is a genome sequence of an individual human. It was produced from approximately 32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2-206 bp), 292,102 heterozygous insertion/deletion events (indels)(1-571 bp), 559,473 homozygous indels (1-82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.

Previously, the majority of the human genome was thought to be 'junk' DNA with no functional purpose. Over the past decade, the field of RNA research has rapidly expanded, with a concomitant increase in the number of non-protein coding RNA (ncRNA) genes identified in this 'junk'. Many of the encoded ncRNAs have already been shown to be essential for a variety of vital functions, and this wealth of annotated human ncRNAs requires standardised naming in order to aid effective communication. The HUGO Gene Nomenclature Committee (HGNC) is the only organisation authorised to assign standardised nomenclature to human genes. Of the 30,000 approved gene symbols currently listed in the HGNC database (http://www.genenames.org/search), the majority represent protein-coding genes; however, they also include pseudogenes, phenotypic loci and some genomic features. In recent years the list has also increased to include almost 3,000 named human ncRNA genes. HGNC is actively engaging with the RNA research community in order to provide unique symbols and names for each sequence that encodes an ncRNA. Most of the classical small ncRNA genes have now been provided with a unique nomenclature, and work on naming the long (>200 nucleotides) non-coding RNAs (lncRNAs) is ongoing.

Telomerase is a ribonucleoprotein complex that replicates the 3' ends of linear chromosomes by successive additions of telomere repeat DNA. The telomerase holoenzyme contains two essential components for catalysis, a telomerase reverse transcriptase (TERT) and telomerase RNA (TER). The TER includes a template for telomere repeat synthesis as well as other domains required for function. We report the solution structure of the wild-type minimal conserved human TER pseudoknot refined with an extensive set of RDCs, and a detailed analysis of the effect of the bulge U177 on pseudoknot structure, dynamics analyzed by RDC and 13C relaxation measurements, and base pair stability. The overall structure of PKWT is highly similar to the previously reported DeltaU177 pseudoknot (PKDU) that has a deletion of a conserved bulge U important for catalytic activity. For direct comparison to PKWT, the structure of PKDU was re-refined with a comparable set of RDCs. Both pseudoknots contain a catalytically essential triple helix at the junction of the two stems, including two stem 1-loop 2 minor groove triples, a junction loop 1-loop 2 Hoogsteen base pair, and stem 2-loop 1 major groove U.A-U Watson-Crick-Hoogsteen triples located directly above the bulge U177. However, there are significant differences in the stabilities of base pairs near the bulge and the dynamics of some nucleotides. The stability of the base pairs in stem 2 surrounding the bulge U177 is greatly decreased, with the result that the Watson-Crick pairs in the triple helix begin to unfold before the Hoogsteen pairs, which may affect telomerase assembly and activity. The bulge U is positioned in the minor groove on the face opposite the triple helical interactions, and sterically blocks the A176 2'OH, which has recently been proposed to have a role in catalysis. The bulge U may serve as a hinge providing backbone flexibility in this region.

Loops which are linkers connecting G-strands and supporting the G-tetrad core in G-quadruplex are important for biological roles of G-quadruplexes. TTA loop is a common sequence which mainly resides in human telomeric DNA (hTel) G-quadruplex. A series of molecular dynamics (MD) simulations were carried out to investigate the structural dynamics of TTA loops. We found that (1) the TA base pair formed in TTA loops are very stable, the occupied of all hydrogen bonds are more than 0.95. (2) The TA base pair makes the adjacent G-quartet more stable than others. (3) For the edgewise loop and the diagonal loop, most loop bases are stacking with others, only few bases have considerable freedom. (4) The stabilities of these stacking structures are distinct. Part of the loops, especially TA base pairs, and bases stacking with the G-quartet, maintain certain stable conformations in the simulation, but other parts, like TT and TA stacking structures, are not stable enough. For the first time, spontaneous conformational switches of TTA edgewise loops were observed in our long time MD simulations. (5) For double chain reversal loop, it is really hard to maintain a stable conformation in the long time simulation under present force fields (parm99 and parmbsc0), as it has multiple conformations with similar free energies.

X-chromosome inactivation is a striking example of epigenetic silencing in which expression of the long non-coding RNA XIST initiates the heterochromatinization and silencing of one of the pair of X chromosomes in mammalian females. To understand how the RNA can establish silencing across millions of basepairs of DNA we have modelled the process by inducing expression of XIST from nine different locations in human HT1080 cells.

Human papillomavirus (HPV) causes 5% of all cancers and frequently integrates into host chromosomes. The HPV oncoproteins E6 and E7 are necessary but insufficient for cancer formation, indicating that additional secondary genetic events are required. Here, we investigate potential oncogenic impacts of virus integration. Analysis of 105 HPV-positive oropharyngeal cancers by whole-genome sequencing detects virus integration in 77%, revealing five statistically significant sites of recurrent integration near genes that regulate epithelial stem cell maintenance (i.e., SOX2, TP63, FGFR, MYC) and immune evasion (i.e., CD274). Genomic copy number hyperamplification is enriched 16-fold near HPV integrants, and the extent of focal host genomic instability increases with their local density. The frequency of genes expressed at extreme outlier levels is increased 86-fold within ±150 kb of integrants. Across 95% of tumors with integration, host gene transcription is disrupted via intragenic integrants, chimeric transcription, outlier expression, gene breaking, and/or de novo expression of noncoding or imprinted genes. We conclude that virus integration can contribute to carcinogenesis in a large majority of HPV-positive oropharyngeal cancers by inducing extensive disruption of host genome structure and gene expression.

Sexual reproduction involving meiosis is essential in most eukaryotes. This produces offspring with novel genotypes, both by segregation of parental chromosomes as well as crossovers between homologous chromosomes. A sexual cycle for the opportunistic human pathogenic fungus Aspergillus fumigatus is known, but the genetic consequences of meiosis have remained unknown. Among other Aspergilli, it is known that A. flavus has a moderately high recombination rate with an average of 4.2 crossovers per chromosome pair, whereas A. nidulans has in contrast a higher rate with 9.3 crossovers per chromosome pair. Here, we show in a cross between A. fumigatus strains that they produce an average of 29.9 crossovers per chromosome pair and large variation in total map length across additional strain crosses. This rate of crossovers per chromosome is more than twice that seen for any known organism, which we discuss in relation to other genetic model systems. We validate this high rate of crossovers through mapping of resistance to the laboratory antifungal acriflavine by using standing variation in an undescribed ABC efflux transporter. We then demonstrate that this rate of crossovers is sufficient to produce one of the common multidrug resistant haplotypes found in the cyp51A gene (TR34/L98H) in crosses among parents harboring either of 2 nearby genetic variants, possibly explaining the early spread of such haplotypes. Our results suggest that genomic studies in this species should reassess common assumptions about linkage between genetic regions. The finding of an unparalleled crossover rate in A. fumigatus provides opportunities to understand why these rates are not generally higher in other eukaryotes.

Although many driver mutations are thought to promote carcinogenesis via abnormal splicing, the landscape of splicing-associated variants (SAVs) remains unknown due to the complexity of splicing abnormalities. Here, we developed a statistical framework to systematically identify SAVs disrupting or newly creating splice site motifs and applied it to matched whole-exome and transcriptome sequencing data from 8976 samples across 31 cancer types, generating a catalog of 14,438 SAVs. Such a large collection of SAVs enabled us to characterize their genomic features, underlying mutational processes, and influence on cancer driver genes. In fact, ∼50% of SAVs identified were those disrupting noncanonical splice sites (non-GT-AG dinucleotides), including the third and fifth intronic bases of donor sites, or newly creating splice sites. Mutation signature analysis revealed that tobacco smoking is more strongly associated with SAVs, whereas ultraviolet exposure has less impact. SAVs showed remarkable enrichment of cancer-related genes, and as many as 14.7% of samples harbored at least one SAVs affecting them, particularly in tumor suppressors. In addition to intron retention, whose association with tumor suppressor inactivation has been previously reported, exon skipping and alternative splice site usage caused by SAVs frequently affected tumor suppressors. Finally, we described high-resolution distributions of SAVs along the gene and their splicing outcomes in commonly disrupted genes, including TP53, PIK3R1, GATA3, and CDKN2A, which offers genetic clues for understanding their functional properties. Collectively, our findings delineate a comprehensive portrait of SAVs, novel insights into transcriptional de-regulation in cancer.

Comparative analysis of recent human genome assemblies highlights profound sequence divergence that peaks within polymorphic loci such as centromeres. This raises the question about the adequacy of relying on human reference genomes to accurately analyze sequencing data derived from experimental cell lines. Here, we generated the complete diploid genome assembly for the human retinal epithelial cells (RPE-1), a widely used non-cancer laboratory cell line with a stable karyotype, to use as matched reference for multi-omics sequencing data analysis. Our RPE1v1.0 assembly presents completely phased haplotypes and chromosome-level scaffolds that span centromeres with ultra-high base accuracy (>QV60). We mapped the haplotype-specific genomic variation specific to this cell line including t(Xq;10q), a stable 73.18 Mb duplication of chromosome 10 translocated onto the microdeleted chromosome X telomere t(Xq;10q). Polymorphisms between haplotypes of the same genome reveals genetic and epigenetic variation for all chromosomes, especially at centromeres. The RPE-1 assembly as matched reference genome improves mapping quality of multi-omics reads originating from RPE-1 cells with drastic reduction in alignments mismatches compared to using the most complete human reference to date (CHM13). Leveraging the accuracy achieved using a matched reference, we were able to identify the kinetochore sites at base pair resolution and show unprecedented variation between haplotypes. This work showcases the use of matched reference genomes for multiomics analyses and serves as the foundation for a call to comprehensively assemble experimentally relevant cell lines for widespread application.

Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.

Next-generation sequencing has implicated some risk variants for human spina bifida (SB), but the genome-wide contribution of structural variation to this complex genetic disorder remains largely unknown. We examined copy-number variant (CNV) participation in the genetic architecture underlying SB risk.

Chromosome segregation errors during meiosis result in the formation of aneuploid gametes and are the leading cause of pregnancy loss and birth defects in humans. Proper chromosome segregation requires pairwise associations of maternal and paternal homologous chromosomes. Chiasmata, which are the cytological manifestations of crossovers (COs), provide a physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Although CO-promoting activities ensure a balanced number and position of COs, their identity and mechanism of action in mammals remain understudied. Previous work in yeast and Arabidopsis has shown that Zip2 and Shoc1 are ortholog proteins with an important role in promoting the formation of COs. Our work is the first study in mammals showing the in vivo and in vitro function of mouse and human SHOC1. We show that purified recombinant human SHOC1, an XPF/MUS81 family member, preferentially binds branched DNA molecules but apparently lacks in vitro endonuclease activity, despite its conserved ERCC4-(HhH)2 core structure. Cytological observations suggest that initial steps of recombination are normal in a majority of spermatocytes from SHOC1 hypomorphic mice. However, late stages of recombination appear abnormal, as chromosomal localization of MLH1 is reduced. In agreement, chiasma formation is reduced, and cells arrest at metaphase I with a few lagging chromosomes and subsequent apoptosis. This analysis of SHOC1-deficient mice and the selective localization of SHOC1 to a subset of recombination sites show that SHOC1 acts at key mid-stage steps of the CO formation process. The formation of chromosome axial elements and homologous pairing are apparently normal, but synapsis is altered with SYCP1 frequently failing to extend the full length of the chromosome axes. Finally, we describe that SHOC1 interacts with TEX11, another protein important for the formation of COs, connecting SHOC1 to chromosome axis and structure.

Viruses need to hijack the translational machinery of the host cell for a productive infection to happen. However, given the dynamic landscape of tRNA pools among tissues, it is unclear whether different viruses infecting different tissues have adapted their codon usage toward their tropism. Here, we collect the coding sequences of 502 human-infecting viruses and determine that tropism explains changes in codon usage. Using the tRNA abundances across 23 human tissues from The Cancer Genome Atlas (TCGA), we build an in silico model of translational efficiency that validates the correspondence of the viral codon usage with the translational machinery of their tropism. For instance, we detect that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is specifically adapted to the upper respiratory tract and alveoli. Furthermore, this correspondence is specifically defined in early viral proteins. The observed tissue-specific translational efficiency could be useful for the development of antiviral therapies and vaccines.

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5'-UUAGGG-3' repeats near their 3'-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5'-UUAGGG-3' repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

Plasmodium species causing malaria in humans are not monophyletic, sharing common ancestors with nonhuman primate parasites. Plasmodium gonderi is one of the few known Plasmodium species infecting African old-world monkeys that are not found in apes. This study reports a de novo assembled P. gonderi genome with complete chromosomes. The P. gonderi genome shares codon usage, syntenic blocks, and other characteristics with the human parasites Plasmodium ovale s.l. and Plasmodium malariae, also of African origin, and the human parasite Plasmodium vivax and species found in nonhuman primates from Southeast Asia. Using phylogenetically aware methods, newly identified syntenic blocks were found enriched with conserved metabolic genes. Regions outside those blocks harbored genes encoding proteins involved in the vertebrate host-Plasmodium relationship undergoing faster evolution. Such genome architecture may have facilitated colonizing vertebrate hosts. Phylogenomic analyses estimated the common ancestor between P. vivax and an African ape parasite P. vivax-like, within the Asian nonhuman primates parasites clade. Time estimates incorporating P. gonderi placed the P. vivax and P. vivax-like common ancestor in the late Pleistocene, a time of active migration of hominids between Africa and Asia. Thus, phylogenomic and time-tree analyses are consistent with an Asian origin for P. vivax and an introduction of P. vivax-like into Africa. Unlike other studies, time estimates for the clade with Plasmodium falciparum, the most lethal human malaria parasite, coincide with their host species radiation, African hominids. Overall, the newly assembled genome presented here has the quality to support comparative genomic investigations in Plasmodium.

The structural maintenance of chromosomes (SMC) proteins constitute the core of critical complexes involved in structural organization of chromosomes. In yeast, the Smc5/6 complex is known to mediate repair of DNA breaks and replication of repetitive genomic regions, including ribosomal DNA loci and telomeres. In mammalian cells, which have diverse genome structure and scale from yeast, the Smc5/6 complex has also been implicated in DNA damage response, but its further function in unchallenged conditions remains elusive. In this study, we addressed the behavior and function of Smc5/6 during the cell cycle. Chromatin fractionation, immunofluorescence, and live-cell imaging analyses indicated that Smc5/6 associates with chromatin during interphase but largely dissociates from chromosomes when they condense in mitosis. Depletion of Smc5 and Smc6 resulted in aberrant mitotic chromosome phenotypes that were accompanied by the abnormal distribution of topoisomerase IIα (topo IIα) and condensins and by chromosome segregation errors. Importantly, interphase chromatin structure indicated by the premature chromosome condensation assay suggested that Smc5/6 is required for the on-time progression of DNA replication and subsequent binding of topo IIα on replicated chromatids. These results indicate an essential role of the Smc5/6 complex in processing DNA replication, which becomes indispensable for proper sister chromatid assembly in mitosis.

Phytophthora root rot (PRR) caused by Phytophthora sojae is a major soybean disease that causes severe economic losses worldwide. Using soybean cultivars carrying a Rps resistance gene is the most effective strategy for controlling this disease. We previously detected a novel Phytophthora resistance gene, RpsZS18, on chromosome 2 of the soybean cultivar Zaoshu18. The aim of the present study was to identify and finely map RpsZS18. We used 232 F2:3 families generated from a cross between Zaoshu18 (resistant) and Williams (susceptible) as the mapping population. Simple sequence repeat (SSR) markers distributed on chromosome 2 were used to map RpsZS18. First, 12 SSR markers linked with RpsZS18 were identified by linkage analyses, including two newly developed SSR markers, ZCSSR33 and ZCSSR46, that flanked the gene at distances of 0.9 and 0.5 cM, respectively. Second, PCR-based InDel markers were developed based on sequence differences between the two parents and used to further narrow down the mapping region of RpsZS18 to 71.3 kb. Third, haplotype analyses were carried out in the RpsZS18 region using 14 soybean genotypes with whole-genome resequencing. We detected six genes with unique haplotype sequences in Zaoshu18. Finally, quantitative real-time PCR assays of the six genes revealed an EF-hand calcium-binding domain containing protein encoding gene (Glyma.02g245700), a pfkB carbohydrate kinase encoding gene (Glyma.02g245800), and a gene with no functional annotation (Glyma.02g246300), are putative candidate PRR resistance genes. This study provides useful information for breeding P. sojae-resistant soybean cultivars.

Common variable immunodeficiency disorder (CVID) is the most common symptomatic primary immunodeficiency in adults, characterized by B-cell abnormalities and inadequate antibody response. CVID patients have considerable autoimmune comorbidity and we therefore hypothesized that genetic susceptibility to CVID may overlap with autoimmune disorders. Here, in the largest genetic study performed in CVID to date, we compare 778 CVID cases with 10,999 controls across 123,127 single-nucleotide polymorphisms (SNPs) on the Immunochip. We identify the first non-HLA genome-wide significant risk locus at CLEC16A (rs17806056, P=2.0 × 10(-9)) and confirm the previously reported human leukocyte antigen (HLA) associations on chromosome 6p21 (rs1049225, P=4.8 × 10(-16)). Clec16a knockdown (KD) mice showed reduced number of B cells and elevated IgM levels compared with controls, suggesting that CLEC16A may be involved in immune regulatory pathways of relevance to CVID. In conclusion, the CLEC16A associations in CVID represent the first robust evidence of non-HLA associations in this immunodeficiency condition.