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Next-generation sequencing (NGS) is a powerful tool for detecting and investigating viral pathogens; however, analysis and management of the enormous amounts of data generated from these technologies remains a challenge. Here, we present VPipe (the Viral NGS Analysis Pipeline and Data Management System), an automated bioinformatics pipeline optimized for whole-genome assembly of viral sequences and identification of diverse species. VPipe automates the data quality control, assembly, and contig identification steps typically performed when analyzing NGS data. Users access the pipeline through a secure web-based portal, which provides an easy-to-use interface with advanced search capabilities for reviewing results. In addition, VPipe provides a centralized system for storing and analyzing NGS data, eliminating common bottlenecks in bioinformatics analyses for public health laboratories with limited on-site computational infrastructure. The performance of VPipe was validated through the analysis of publicly available NGS data sets for viral pathogens, generating high-quality assemblies for 12 data sets. VPipe also generated assemblies with greater contiguity than similar pipelines for 41 human respiratory syncytial virus isolates and 23 SARS-CoV-2 specimens.

Invasive meningococcal disease is mainly caused by Neisseria meningitidis serogroups A, B, C, X, W, and Y. The serogroup is typically determined by slide agglutination serogrouping (SASG) and real-time PCR (RT-PCR). We describe a whole-genome sequencing (WGS)-based method to characterize the capsule polysaccharide synthesis (cps) locus, classify N. meningitidis serogroups, and identify mechanisms for nongroupability using 453 isolates from a global strain collection. We identified novel genomic organizations within functional cps loci, consisting of insertion sequence (IS) elements in unique positions that did not disrupt the coding sequence. Genetic mutations (partial gene deletion, missing genes, IS insertion, internal stop, and phase-variable off) that led to nongroupability were identified. The results of WGS and SASG were in 91% to 100% agreement for all serogroups, while the results of WGS and RT-PCR showed 99% to 100% agreement. Among isolates determined to be nongroupable by WGS (31 of 453), the results of all three methods agreed 100% for those without a capsule polymerase gene. However, 61% (WGS versus SASG) and 36% (WGS versus RT-PCR) agreements were observed for the isolates, particularly those with phase variations or internal stops in cps loci, which warrant further characterization by additional tests. Our WGS-based serogrouping method provides comprehensive characterization of the N. meningitidis capsule, which is critical for meningococcal surveillance and outbreak investigations.

In Drosophila, dosage compensation is mediated by the MSL complex, which binds numerous sites on the X chromosome in males and enhances the transcriptional rate of a substantial number of X-linked genes. We have determined that topoisomerase II (Topo II) is enriched on dosage compensated genes, to which it is recruited by association with the MSL complex, in excess of the amount that is present on autosomal genes with similar transcription levels. Using a plasmid model, we show that Topo II is required for proper dosage compensation and that compensated chromatin is topologically different from non-compensated chromatin. This difference, which is not the result of the enhanced transcription level due of X-linked genes and which represents a structural modification intrinsic to the DNA of compensated chromatin, requires the function of Topo II. Our results suggest that Topo II is an integral part of the mechanistic basis of dosage compensation.

Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

Understanding the relationship between genome organization and expression is central to understanding genome function. Closely apposed genes in a head-to-head orientation share the same upstream region and are likely to be coregulated. Here we identify the Drosophila BEAF-32 insulator as a cis regulatory element separating close head-to-head genes with different transcription regulation modes. We then compare the binding landscapes of the BEAF-32 insulator protein in four different Drosophila genomes and highlight the evolutionarily conserved presence of this protein between close adjacent genes. We find that changes in binding of BEAF-32 to sites in the genome of different Drosophila species correlate with alterations in genome organization caused by DNA rearrangements or genome size expansion. The cross-talk between BEAF-32 genomic distribution and genome organization contributes to new gene-expression profiles, which in turn translate into specific and distinct phenotypes. The results suggest a mechanism for the establishment of differences in transcription patterns during evolution.

Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions.

Transcription regulation is mediated by enhancers that bind sequence-specific transcription factors, which in turn interact with the promoters of the genes they control. Here, we show that the JIL-1 kinase is present at both enhancers and promoters of ecdysone-induced Drosophila genes, where it phosphorylates the Ser10 and Ser28 residues of histone H3. JIL-1 is also required for CREB binding protein (CBP)-mediated acetylation of Lys27, a well-characterized mark of active enhancers. The presence of these proteins at enhancers and promoters of ecdysone-induced genes results in the establishment of the H3K9acS10ph and H3K27acS28ph marks at both regulatory sequences. These modifications are necessary for the recruitment of 14-3-3, a scaffolding protein capable of facilitating interactions between two simultaneously bound proteins. Chromatin conformation capture assays indicate that interaction between the enhancer and the promoter is dependent on the presence of JIL-1, 14-3-3, and CBP. Genome-wide analyses extend these conclusions to most Drosophila genes, showing that the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph is a general feature of enhancers and promoters in this organism.

Total serum bilirubin is associated with several clinical outcomes, including cardiovascular disease, diabetes and drug metabolism. We conducted a genome-wide association study in 619 healthy unrelated African Americans in an attempt to replicate reported findings in Europeans and Asians and to identify novel loci influencing total serum bilirubin levels. We analyzed a dense panel of over two million genotyped and imputed SNPs in additive genetic models adjusting for age, sex, and the first two significant principal components from the sample covariance matrix of genotypes. Thirty-nine SNPs spanning a 78 kb region within the UGT1A1 displayed P-values5 × 10(-8). The lowest P-value was 1.7 × 10(-22) for SNP rs887829. None of SNPs in the UGT1A1 remained statistically significant in conditional association analyses that adjusted for rs887829. In addition, SNP rs10929302 located in phenobarbital response enhancer module was significantly associated with bilirubin level with a P-value of 1.37 × 10(-11); this enhancer module is believed to have a critical role in phenobarbital treatment of hyperbilirubinemia. Interestingly, the lead SNP, rs887829, is in strong linkage disequilibrium (LD) (r(2)≥0.74) with rs10929302. Taking advantage of the lower LD and shorter haplotypes in African-ancestry populations, we identified rs887829 as a more refined proxy for the causative variant influencing bilirubin levels. Also, we replicated the reported association between variants in SEMA3C and bilirubin levels. In summary, UGT1A1 is a major locus influencing bilirubin levels and the results of this study promise to contribute to understanding of the etiology and treatment of hyperbilirubinaemia in African-ancestry populations.

Insulators are multi-protein-DNA complexes thought to affect gene expression by mediating inter- and intra-chromosomal interactions. Drosophila insulators contain specific DNA binding proteins plus common components, such as CP190, that facilitate these interactions. Here we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to the DNA in order to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve proper gene expression in response to different stimuli.

Aims/hypothesis Chronically elevated blood glucose (hyperglycaemia) is the primary indicator of type 2 diabetes, which has a prevalence that varies considerably by ethnicity in the USA, with African-Americans disproportionately affected. Genome-wide association studies (GWASs) have significantly enhanced our understanding of the genetic basis of diabetes and related traits, including fasting plasma glucose (FPG). However, the majority of GWASs have been conducted in populations of European ancestry. Thus, it is important to conduct replication analyses in populations with non-European ancestry to identify shared loci associated with FPG across populations. Methods We used data collected from non-diabetic unrelated African-American individuals (n = 927) who participated in the Howard University Family Study to attempt to replicate previously published GWASs of FPG. Of the 29 single nucleotide polymorphisms (SNPs) previously reported, we directly tested 20 in this study. In addition to the direct test, we queried a 500 kb window centred on all 29 reported SNPs for local replication of additional markers in linkage disequilibrium (LD). Results Using direct SNP and LD-based comparisons, we replicated multiple SNPs previously associated with FPG and strongly associated with type 2 diabetes in populations with European ancestry. The replicated SNPs included those in or near TCF7L2, SLC30A8, G6PC2, MTNR1B, DGKB-TMEM195 and GCKR. We also replicated additional variants in LD with the reported SNPs in ZMAT4 and adjacent to IRS1. Conclusions/interpretation We identified multiple GWAS variants for FPG in our cohort of African-Americans. Using an LD-based strategy we also identified SNPs not previously reported, demonstrating the utility of using diverse populations for replication analysis. Electronic supplementary material The online version of this article (doi:10.1007/s00125-010-2002-7) contains supplementary material, which is available to authorised users.

Background Benign ethnic neutropenia (BEN) is a hematologic condition associated with people of African ancestry and specific Middle Eastern ethnic groups. Prior genetic association studies in large population showed that rs2814778 in Duffy Antigen Receptor for Chemokines (DARC) gene, specifically DARC null red cell phenotype, was associated with BEN. However, the mechanism of this red cell phenotype leading to low white cell count remained elusive. Methods We conducted an extreme phenotype design genome-wide association study (GWAS), analyzed ~16 million single nucleotide polymorphisms (SNP) in 1,178 African-Americans individuals from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study and replicated from 819 African-American participants in the Atherosclerosis Risk in Communities (ARIC) study. Conditional analyses on rs2814778 were performed to identify additional association signals on chromosome 1q22. In a separate cohort of healthy individuals with and without BEN, whole genome gene expression from peripheral blood neutrophils were analyzed for DARC. Results We confirmed that rs2814778 in DARC was associated with BEN (p = 4.09×10-53). Conditioning on rs2814778 abolished other significant chromosome 1 associations. Inflammatory cytokines (IL-2, 6, and 10) in participants in the Howard University Family Study (HUFS) and Multi-Ethnic Study in Atherosclerosis (MESA) showed similar levels in individuals homozygous for the rs2814778 allele compared to others, indicating cytokine sink hypothesis played a minor role in leukocyte homeostasis. Gene expression in neutrophils of individuals with and without BEN was also similar except for low DARC expression in BEN, suggesting normal function. BEN neutrophils had slightly activated profiles in leukocyte migration and hematopoietic stem cell mobilization pathways (expression fold change Conclusions These results in humans support the notion of DARC null erythroid progenitors preferentially differentiating to myeloid cells, leading to activated DARC null neutrophils egressing from circulation to the spleen, and causing relative neutropenia. Collectively, these human data sufficiently explained the mechanism DARC null red cell phenotype causing BEN and further provided a biologic basis that BEN is clinically benign.

Chromatin insulators have been implicated in the regulation of higher-order chromatin structure and may function to compartmentalize the eukaryotic genome into independent domains of gene expression. To test this possibility, we used biochemical and computational approaches to identify gypsy-like genomic-binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein, a component of the gypsy insulator. EMSA and FISH analyses suggest that these are genuine Su(Hw)-binding sites. In addition, functional tests indicate that genomic Su(Hw)-binding sites can inhibit enhancer–promoter interactions and thus function as bona fide insulators. The insulator strength is dependent on the genomic location of the transgene and the number of Su(Hw)-binding sites, with clusters of two to three sites showing a stronger effect than individual sites. These clusters of Su(Hw)-binding sites are located mostly in intergenic regions or in introns of large genes, an arrangement that fits well with their proposed role in the formation of chromatin domains. Taken together, these data suggest that genomic gypsy-like insulators may provide a means for the compartmentalization of the genome within the nucleus.

SummaryAppropriate cell number and organ size in a multicellular organism are determined by coordinated cell growth, proliferation, and apoptosis. Disruption of these processes can cause cancer. Recent studies have identified the Large tumor suppressor (Lats)/Warts (Wts) protein kinase as a key component of a pathway that controls the coordination between cell proliferation and apoptosis. Here we describe growth inhibitory functions for a Mob superfamily protein, termed Mats (Mob as tumor suppressor), in Drosophila. Loss of Mats function results in increased cell proliferation, defective apoptosis, and induction of tissue overgrowth. We show that mats and wts function in a common pathway. Mats physically associates with Wts to stimulate the catalytic activity of the Wts kinase. A human Mats ortholog (Mats1) can rescue the lethality associated with loss of Mats function in Drosophila. As Mats1 is mutated in human tumors, Mats-mediated growth inhibition and tumor suppression is likely conserved in humans.

Receptor tyrosine kinase (RTK) signaling plays an instructive role in cell fate decisions, whereas Notch signaling is often involved in restricting cellular competence for differentiation. Genetic interactions between these two evolutionarily conserved pathways have been extensively documented. The underlying molecular mechanisms, however, are not well understood. Here, we show that Yan, an Ets transcriptional repressor that blocks cellular potential for specification and differentiation [1, 2], is a target of Notch signaling during Drosophila eye development. The Suppressor of Hairless (Su[H]) protein of the Notch pathway is required for activating yan expression, and Su(H) binds directly to an eye-specific yan enhancer in vitro. In contrast, yan expression is repressed by Pointed (Pnt), which is a key component of the RTK pathway. Pnt binds specifically to the yan enhancer and competes with Su(H) for DNA binding. This competition illustrates a potential mechanism for RTK and Notch signals to oppose each other. Thus, yan serves as a common target of Notch/Su(H) and RTK/Pointed signaling pathways during cell fate specification.

Insulin resistance (IR) is a key determinant of type 2 diabetes (T2D) and other metabolic disorders. This genome-wide association study (GWAS) was designed to shed light on the genetic basis of fasting insulin (FI) and IR in 927 non-diabetic African Americans. 5 396 838 single-nucleotide polymorphisms (SNPs) were tested for associations with FI or IR with adjustments for age, sex, body mass index, hypertension status and first two principal components. Genotyped SNPs (n = 12) with P5 × 10(-6) in African Americans were carried forward for de novo genotyping in 570 non-diabetic West Africans. We replicated SNPs in or near SC4MOL and TCERG1L in West Africans. The meta-analysis of 1497 African Americans and West Africans yielded genome-wide significant associations for SNPs in the SC4MOL gene: rs17046216 (P = 1.7 × 10(-8) and 2.9 × 10(-8) for FI and IR, respectively); and near the TCERG1L gene with rs7077836 as the top scoring (P = 7.5 × 10(-9) and 4.9 × 10(-10) for FI and IR, respectively). In silico replication in the MAGIC study (n = 37 037) showed weak but significant association (adjusted P-value of 0.0097) for rs34602777 in the MYO5A gene. In addition, we replicated previous GWAS findings for IR and FI in Europeans for GCKR, and for variants in four T2D loci (FTO, IRS1, KLF14 and PPARG) which exert their action via IR. In summary, variants in/near SC4MOL, and TCERG1L were associated with FI and IR in this cohort of African Americans and were replicated in West Africans. SC4MOL is under-expressed in an animal model of T2D and plays a key role in lipid biosynthesis, with implications for the regulation of energy metabolism, obesity and dyslipidemia. TCERG1L is associated with plasma adiponectin, a key modulator of obesity, inflammation, IR and diabetes.

Insulators are DNA sequences thought to be important for the establishment and maintenance of cell-type specific nuclear architecture. In Drosophila there are several classes of insulators that appear to have unique roles in gene expression. The mechanisms involved in determining and regulating the specific roles of these insulator classes are not understood. Here we report that DNA Topoisomerase II modulates the activity of the Su(Hw) insulator. Downregulation of Topo II by RNAi or mutations in the Top2 gene result in disruption of Su(Hw) insulator function. This effect is mediated by the Mod(mdg4)2.2 protein, which is a unique component of the Su(Hw) insulator complex. Co-immunoprecipitation and yeast two-hybrid experiments show that Topo II and Mod(mdg4)2.2 proteins directly interact. In addition, mutations in Top2 cause a slight decrease of Mod(mdg4)2.2 transcript but have a dramatic effect on Mod(mdg4)2.2 protein levels. In the presence of proteasome inhibitors, normal levels of Mod(mdg4)2.2 protein and its binding to polytene chromosomes are restored. Thus, Topo II is required to prevent Mod(mdg4)2.2 degradation and, consequently, to stabilize Su(Hw) insulator-mediated chromatin organization.

Background The study of human genetic variation has been advanced by research such as genome-wide association studies, which aim to identify variants associated with common, complex diseases and traits. Significant strides have already been made in gleaning information on susceptibility, treatment, and prevention of a number of disorders. However, as genetic researchers continue to uncover underlying differences between individuals, there is growing concern that observed population-level differences will be inappropriately generalized as inherent to particular racial or ethnic groups and potentially perpetuate negative stereotypes. Discussion We caution that imprecision of language when conveying research conclusions, compounded by the potential distortion of findings by the media, can lead to the stigmatization of racial and ethnic groups. Summary It is essential that the scientific community and with those reporting and disseminating research findings continue to foster a socially responsible dialogue about genetic variation and human difference.

Insulators are protein-bound DNA elements that are thought to play a role in chromatin organization and the regulation of gene expression by mediating intra- and interchromosomal interactions. Suppressor of Hair-wing [Su(Hw)] and Drosophila CTCF (dCTCF) insulators are found at distinct loci throughout the Drosophila melanogaster genome and function by recruiting an additional protein, Centrosomal Protein 190 (CP190). We performed chromatin immunoprecipitation (ChIP) and microarray analysis (ChIP–chip) experiments with whole-genome tiling arrays to compare Su(Hw), dCTCF, boundary element-associated factor (BEAF), and CP190 localization on DNA in two different cell lines and found evidence that BEAF is a third subclass of CP190-containing insulators. The DNA-binding proteins Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization. Notably, cell line-specific localization sites for all three DNA-binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression. These findings suggest a model in which insulator subclasses may have distinct functions that together organize the genome in a cell type-specific manner, resulting in differential regulation of gene expression.

Background DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters. Results Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined. Conclusion The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.

Yan is a nuclear DNA-binding protein that acts as a general inhibitor of cellular differentiation and proliferation in Drosophila melanogaster. The genetic and biochemical mechanisms required for regulating Yan protein function are well understood, however, the molecular mechanism of yan gene transcriptional regulation has not been fully elucidated. Here we show that the dynamic expression of the yan gene is specified by distinct spatial and temporal cis-acting regulatory elements in embryos and larval tissues. Each of these distinct elements is thus capable of replicating vital aspects of endogenous yan gene expression.

In order to eliminate health disparities in the United States, more efforts are needed to address the breadth of social issues directly contributing to the healthy divide observed across racial and ethnic groups. Socioeconomic status, education, and the environment are intimately linked to health outcomes. However, with the tremendous advances in technology and increased investigation into human genetic variation, genomics is poised to play a valuable role in bolstering efforts to find new treatments and preventions for chronic conditions and diseases that disparately affect certain ethnic groups. Promising studies focused on understanding the genetic underpinnings of diseases such as prostate cancer or beta-blocker treatments for heart failure are illustrative of the positive contribution that genomics can have on improving minority health.