Systematic identification of factors involved in the silencing of germline genes in mouse embryonic stem cells.

In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation deposition at germline gene promoters for long term repression. Our study provides a global view of the mechanisms and novel factors required for silencing germline genes in embryonic stem cells.

A comparative methylome analysis reveals conservation and divergence of DNA methylation patterns and functions in vertebrates.

BACKGROUND: Cytosine DNA methylation is a heritable epigenetic mark present in most eukaryotic groups. While the patterns and functions of DNA methylation have been extensively studied in mouse and human, their conservation in other vertebrates remains poorly explored. In this study, we interrogated the distribution and function of DNA methylation in primary fibroblasts of seven vertebrate species including bio-medical models and livestock species (human, mouse, rabbit, dog, cow, pig, and chicken). RESULTS: Our data highlight both divergence and conservation of DNA methylation patterns and functions. We show that the chicken genome is hypomethylated compared to other vertebrates. Furthermore, compared to mouse, other species show a higher frequency of methylation of CpG-rich DNA. We reveal the conservation of large unmethylated valleys and patterns of DNA methylation associated with X-chromosome inactivation through vertebrate evolution and make predictions of conserved sets of imprinted genes across mammals. Finally, using chemical inhibition of DNA methylation, we show that the silencing of germline genes and endogenous retroviruses (ERVs) are conserved functions of DNA methylation in vertebrates. CONCLUSIONS: Our data highlight conserved properties of DNA methylation in vertebrate genomes but at the same time point to differences between mouse and other vertebrate species.

De novo germline mutation in the dual specificity phosphatase 10 gene accelerates autoimmune diabetes.

Insulin-dependent or type 1 diabetes (T1D) is a polygenic autoimmune disease. In humans, more than 60 loci carrying common variants that confer disease susceptibility have been identified by genome-wide association studies, with a low individual risk contribution for most variants excepting those of the major histocompatibility complex (MHC) region (40 to 50% of risk); hence the importance of missing heritability due in part to rare variants. Nonobese diabetic (NOD) mice recapitulate major features of the human disease including genetic aspects with a key role for the MHC haplotype and a series of Idd loci. Here we mapped in NOD mice rare variants arising from genetic drift and significantly impacting disease risk. To that aim we established by selective breeding two sublines of NOD mice from our inbred NOD/Nck colony exhibiting a significant difference in T1D incidence. Whole-genome sequencing of high (H)- and low (L)-incidence sublines (NOD/NckH and NOD/NckL) revealed a limited number of subline-specific variants. Treating age of diabetes onset as a quantitative trait in automated meiotic mapping (AMM), enhanced susceptibility in NOD/NckH mice was unambiguously attributed to a recessive missense mutation of Dusp10, which encodes a dual specificity phosphatase. The causative effect of the mutation was verified by targeting Dusp10 with CRISPR-Cas9 in NOD/NckL mice, a manipulation that significantly increased disease incidence. The Dusp10 mutation resulted in islet cell down-regulation of type I interferon signature genes, which may exert protective effects against autoimmune aggression. De novo mutations akin to rare human susceptibility variants can alter the T1D phenotype.

Genome assembly of a Mesoamerican derived variety of lima bean: a foundational cultivar in the Mid-Atlantic USA.

Lima bean, Phaseolus lunatus, is closely related to common bean and is high in fiber and protein, with a low glycemic index. Lima bean is widely grown in the state of Delaware, where late summer and early fall weather are conducive to pod production. The same weather conditions also promote diseases such as pod rot and downy mildew, the latter of which has caused previous epidemics. A better understanding of the genes underlying resistance to this and other pathogens is needed to keep this industry thriving in the region. Our current study sought to sequence, assemble, and annotate a commercially available cultivar called Bridgeton, which could then serve as a reference genome, a basis of comparison to other Phaseolus taxa, and a resource for the identification of potential resistance genes. Combined efforts of sequencing, linkage, and comparative analysis resulted in a 623 Mb annotated assembly for lima bean, as well as a better understanding of an evolutionarily dynamic resistance locus in legumes.

E2F6 initiates stable epigenetic silencing of germline genes during embryonic development.

In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse development.

Technical Note: Comparison of the internal target volume (ITV) contours and dose calculations on 4DCT, average CBCT, and 4DCBCT imaging for lung stereotactic body radiation therapy (SBRT).

PURPOSE: To investigate the differences between internal target volumes (ITVs) contoured on the simulation 4DCT and daily 4DCBCT images for lung cancer patients treated with stereotactic body radiotherapy (SBRT) and determine the dose delivered on 4D planning technique. METHODS: For nine patients, 4DCBCTs were acquired before each fraction to assess tumor motion. An ITV was contoured on each phase of the 4DCBCT and a union of the 10 ITVs was used to create a composite ITV. Another ITV was drawn on the average 3DCBCT (avgCBCT) to compare with current clinical practice. The Dice coefficient, Hausdorff distance, and center of mass (COM) were averaged over four fractions to compare the ITVs contoured on the 4DCT, avgCBCT, and 4DCBCT for each patient. Planning was done on the average CT, and using the online registration, plans were calculated on each phase of the 4DCBCT and on the avgCBCT. Plan dose calculations were tested by measuring ion chamber dose in the CIRS lung phantom. RESULTS: The Dice coefficients were similar for all three comparisons: avgCBCT-to-4DCBCT (0.7 ± 0.1), 4DCT-to-avgCBCT (0.7 ± 0.1), and 4DCT-to-4DCBCT (0.7 ± 0.1); while the mean COM differences were also comparable (2.6 ± 2.2mm, 2.3 ± 1.4mm, and 3.1 ± 1.1mm, respectively). The Hausdorff distances for the comparisons with 4DCBCT (8.2 ± 2.9mm and 8.1 ± 3.2mm) were larger than the comparison without (6.5 ± 2.5mm). The differences in ITV D95% between the treatment plan and avgCBCT calculations were 4.3 ± 3.0% and -0.5 ± 4.6%, between treatment plan and 4DCBCT plans, respectively, while the ITV V100% coverages were 99.0 ± 1.9% and 93.1 ± 8.0% for avgCBCT and 4DCBCT, respectively. CONCLUSION: There is great potential for 4DCBCT to evaluate the extent of tumor motion before treatment, but image quality challenges the clinician to consistently delineate lung target volumes.

Genome-wide analysis in the mouse embryo reveals the importance of DNA methylation for transcription integrity.

Mouse embryos acquire global DNA methylation of their genome during implantation. However the exact roles of DNA methyltransferases (DNMTs) in embryos have not been studied comprehensively. Here we systematically analyze the consequences of genetic inactivation of Dnmt1, Dnmt3a and Dnmt3b on the methylome and transcriptome of mouse embryos. We find a strict division of function between DNMT1, responsible for maintenance methylation, and DNMT3A/B, solely responsible for methylation acquisition in development. By analyzing severely hypomethylated embryos, we uncover multiple functions of DNA methylation that is used as a mechanism of repression for a panel of genes including not only imprinted and germline genes, but also lineage-committed genes and 2-cell genes. DNA methylation also suppresses multiple retrotransposons and illegitimate transcripts from cryptic promoters in transposons and gene bodies. Our work provides a thorough analysis of the roles of DNA methyltransferases and the importance of DNA methylation for transcriptome integrity in mammalian embryos.

Loss of Apc Rapidly Impairs DNA Methylation Programs and Cell Fate Decisions in Lgr5+ Intestinal Stem Cells.

Colorectal cancer initiation and progression result from the accumulation of genetic and epigenetic alterations. Although aberrant gene expression and DNA methylation profiles are considered hallmarks of colorectal cancer development, the precise timing at which these are produced during tumor establishment remains elusive. Here we investigated the early transcriptional and epigenetic changes induced by adenomatous polyposis coli (Apc) inactivation in intestinal crypts. Hyperactivation of the Wnt pathway via Apc inactivation in crypt base columnar intestinal stem cells (ISC) led to their rapid accumulation driven by an impaired molecular commitment to differentiation, which was associated with discrete alterations in DNA methylation. Importantly, inhibiting the enzymes responsible for de novo DNA methylation restored the responsiveness of Apc-deficient intestinal organoids to stimuli regulating the proliferation-to-differentiation transition in ISC. This work reveals that early DNA methylation changes play critical roles in the establishment of the impaired fate decision program consecutive to Apc loss of function. SIGNIFICANCE: This study demonstrates the functional impact of changes in DNA methylation to determine the colorectal cancer cell phenotype following loss of Apc function.

Distinct oncogenes drive different genome and epigenome alterations in human mammary epithelial cells.

Molecular subtypes of breast cancer are defined on the basis of gene expression and genomic/epigenetic pattern differences. Different subtypes are thought to originate from distinct cell lineages, but the early activation of an oncogene could also play a role. It is difficult to discriminate the respective inputs of oncogene activation or cell type of origin. In this work, we wished to determine whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. To this aim, we transduced shp53 immortalized HMECs in parallel with the CCNE1, WNT1 and RASv12 oncogenes which activate distinct oncogenic pathways and characterized them at sequential stages of transformation for changes in their genetic and epigenetic profiles. We show that initial activation of CCNE1, WNT1 and RASv12, in shp53 HMECs results in different and reproducible changes in mRNA and micro-RNA expression, copy number alterations (CNA) and DNA methylation profiles. Noticeably, HMECs transformed by RAS bore very specific profiles of CNAs and DNA methylation, clearly distinct from those shown by CCNE1 and WNT1 transformed HMECs. Genes impacted by CNAs and CpG methylation in the RAS and the CCNE1/WNT1 clusters showed clear differences, illustrating the activation of distinct pathways. Our data show that early activation of distinct oncogenic pathways leads to active adaptive events resulting in specific sets of CNAs and DNA methylation changes. We, thus, propose that activation of different oncogenes could have a role in reshaping the genetic landscape of breast cancer subtypes.

Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries.

BACKGROUND: Targeted resequencing with high-throughput sequencing (HTS) platforms can be used to efficiently interrogate the genomes of large numbers of individuals. A critical issue for research and applications using HTS data, especially from long-read platforms, is error in base calling arising from technological limits and bioinformatic algorithms. We found that the community standard long amplicon analysis (LAA) module from Pacific Biosciences is prone to substantial bioinformatic errors that raise concerns about findings based on this pipeline, prompting the need for a new method. RESULTS: A single molecule real-time (SMRT) sequencing-error correction and assembly pipeline, C3S-LAA, was developed for libraries of pooled amplicons. By uniquely leveraging the structure of SMRT sequence data (comprised of multiple low quality subreads from which higher quality circular consensus sequences are formed) to cluster raw reads, C3S-LAA produced accurate consensus sequences and assemblies of overlapping amplicons from single sample and multiplexed libraries. In contrast, despite read depths in excess of 100X per amplicon, the standard long amplicon analysis module from Pacific Biosciences generated unexpected numbers of amplicon sequences with substantial inaccuracies in the consensus sequences. A bootstrap analysis showed that the C3S-LAA pipeline per se was effective at removing bioinformatic sources of error, but in rare cases a read depth of nearly 400X was not sufficient to overcome minor but systematic errors inherent to amplification or sequencing. CONCLUSIONS: C3S-LAA uses a divide and conquer processing algorithm for SMRT amplicon-sequence data that generates accurate consensus sequences and local sequence assemblies. Solving the confounding bioinformatic source of error in LAA allowed for the identification of limited instances of errors due to DNA amplification or sequencing of homopolymeric nucleotide tracts. For research and development in genomics, C3S-LAA allows meaningful conclusions and biological inferences to be made from accurately polished sequence output.

Patient Navigation for Patients Frequently Visiting the Emergency Department: A Randomized, Controlled Trial.

BACKGROUND: Emergency department (ED) superutilizers (patients with five or more visits/year) comprise only 5% of the patients seen yet comprise 25% of total ED visits. Although the reasons for this are multifactorial, the cost to the patient and the community is exceedingly high. The cost is not just monetary; care of these patients is inappropriately fragmented and their presence in the ED may contribute to overcrowding affecting the community's emergency readiness. Previous studies using staff trained to help patients navigate their care options have had conflicting results. OBJECTIVES: The objective was to determine whether a trained patient navigator (PN) can reduce ED use and costs in superutilizers over a 1-year period. METHODS: Superutilizers were enrolled in a prospective randomized controlled clinical trial. Patients were randomized into the treatment arm and met with a PN who reviewed their diagnosis and associated care plan and identified proper primary care services and community resources for follow-up. The remaining control group was provided standard care. Both groups were given a follow-up call and survey by the PN within 7 days of their visit who assessed primary care follow-up and patient satisfaction using a 4-point Likert scale. After 12 months, the patients' return ED visits and ED costs were compared to the year prior and primary care compliance and satisfaction were measured using Student's t-tests with Bonferroni correction or Mann-Whitney U-tests. RESULTS: A total of 282 patients were enrolled (148 in navigation treatment group, 134 controls). Patients were similarly matched in age, race, sex, insurance, and chief complaints. Overall ED visits decreased during the 12-month study period, compared to the 12 months prior to enrollment (2,249 visits prior to 2,050 visits during study period, -8.8%). There was a greater decrease in ED visits from the preenrollment year to postenrollment year in the treatment group (1,148 visits to 996 visits, -13.2%) compared to the control group (1,101 visits to 1,054 visits, -4.3%; p < 0.05). Overall health care costs (ED and hospital) for all 282 patients decreased in the year after compared to the 12 months prior to enrollment ($3.9M to $3.1M) with a greater decrease in the navigation treatment group (-26.6%) compared to the control group (-17.5%). Patient surveys found no difference in patient satisfaction in the pre- and postenrollment periods but there was an increase in primary care physician (PCP) use over the 12-month follow-up period in the treatment group (6.42 visits/patient) compared to the control group (4.07 visits/patient; p < 0.05). CONCLUSION: Our data showed that the overall number of return ED visits and costs did decrease for both groups, potentially inferring a placebo effect for the use of a PN; however, the decrease in ED visits and costs were greater in the treatment group. One-year follow-up noted an increase in PCP visits in the navigation group. Use of a PN may be cost-effective.

ThermoAlign: a genome-aware primer design tool for tiled amplicon resequencing.

Isolating and sequencing specific regions in a genome is a cornerstone of molecular biology. This has been facilitated by computationally encoding the thermodynamics of DNA hybridization for automated design of hybridization and priming oligonucleotides. However, the repetitive composition of genomes challenges the identification of target-specific oligonucleotides, which limits genetics and genomics research on many species. Here, a tool called ThermoAlign was developed that ensures the design of target-specific primer pairs for DNA amplification. This is achieved by evaluating the thermodynamics of hybridization for full-length oligonucleotide-template alignments - thermoalignments - across the genome to identify primers predicted to bind specifically to the target site. For amplification-based resequencing of regions that cannot be amplified by a single primer pair, a directed graph analysis method is used to identify minimum amplicon tiling paths. Laboratory validation by standard and long-range polymerase chain reaction and amplicon resequencing with maize, one of the most repetitive genomes sequenced to date (≈85% repeat content), demonstrated the specificity-by-design functionality of ThermoAlign. ThermoAlign is released under an open source license and bundled in a dependency-free container for wide distribution. It is anticipated that this tool will facilitate multiple applications in genetics and genomics and be useful in the workflow of high-throughput targeted resequencing studies.

EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos.

The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2(-/-) embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development.

A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement.

BACKGROUND: Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. METHODS: We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. RESULTS: We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. CONCLUSIONS: We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. TRIAL REGISTRATION NUMBERS: NCT01746121 and NCT02397824.

Sensitivity and variability of Presage dosimeter formulations in sheet form with application to SBRT and SRS QA.

PURPOSE: To measure sensitivity and stability of the Presage dosimeter in sheet form for various chemical concentrations over a range of clinical photon energies and examine its use for stereotactic body radiation therapy (SBRT) and stereotactic radiosurgery (SRS) QA. METHODS: Presage polymer dosimeters were formulated to investigate and optimize their sensitivity and stability. The dosimeter is composed of clear polyurethane base, leucomalachite green (LMG) reporting dye, and bromoform radical initiator in 0.9-1.0 mm thick sheets. The chemicals are mixed together for 2 min, cast in an aluminum mold, and left to cure at 60 psi for a minimum of two days. Dosimeter response was characterized at energies Co-60, 6 MV, 10 MV flattening-filter free, 15 MV, 50 kVp (mean 19.2 keV), and Ir-192. The dosimeters were scanned by a Microtek Scanmaker i800 at 300 dpi, 2(16) bit depth per color channel. Red component images were analyzed with ImageJ and rit. SBRT QA was done with gamma analysis tolerances of 2% and 2 mm DTA. RESULTS: The sensitivity of the Presage dosimeter increased with increasing concentration of bromoform. Addition of tin catalyst decreased curing time and had negligible effect on sensitivity. LMG concentration should be at least as high as the bromoform, with ideal concentration being 2% wt. Gamma Knife SRS QA measurements of relative output and profile widths were within 2% of manufacturer's values validated at commissioning, except the 4 mm collimator relative output which was within 3%. The gamma pass rate of Presage with SBRT was 73.7%, compared to 93.1% for EBT2 Gafchromic film. CONCLUSIONS: The Presage dosimeter in sheet form was capable of detecting radiation over all tested photon energies and chemical concentrations. The best sensitivity and photostability of the dosimeter were achieved with 2.5% wt. LMG and 8.2% wt. bromoform. Scanner used should not emit any UV radiation as it will expose the dosimeter, as with the Epson 10000 XL scanner. Presage dosimeter in this form was sensitive enough for use in SRS and SBRT QA. The lower gamma pass rate for Presage compared to Gafchromic film can be attributed to the simple equipment used in the fabrication process, which limited the dosimeter's sensitivity uniformity by agglomeration of air bubbles in the material, nonuniform concentration of chemicals throughout the material, and thickness variations. This demands improvements in mixing tools and molds.

Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing.

BACKGROUND: Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation. METHODS: We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases. RESULTS: We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients' clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders. CONCLUSIONS: With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism.

VIROME: a standard operating procedure for analysis of viral metagenome sequences.

One consistent finding among studies using shotgun metagenomics to analyze whole viral communities is that most viral sequences show no significant homology to known sequences. Thus, bioinformatic analyses based on sequence collections such as GenBank nr, which are largely comprised of sequences from known organisms, tend to ignore a majority of sequences within most shotgun viral metagenome libraries. Here we describe a bioinformatic pipeline, the Viral Informatics Resource for Metagenome Exploration (VIROME), that emphasizes the classification of viral metagenome sequences (predicted open-reading frames) based on homology search results against both known and environmental sequences. Functional and taxonomic information is derived from five annotated sequence databases which are linked to the UniRef 100 database. Environmental classifications are obtained from hits against a custom database, MetaGenomes On-Line, which contains 49 million predicted environmental peptides. Each predicted viral metagenomic ORF run through the VIROME pipeline is placed into one of seven ORF classes, thus, every sequence receives a meaningful annotation. Additionally, the pipeline includes quality control measures to remove contaminating and poor quality sequence and assesses the potential amount of cellular DNA contamination in a viral metagenome library by screening for rRNA genes. Access to the VIROME pipeline and analysis results are provided through a web-application interface that is dynamically linked to a relational back-end database. The VIROME web-application interface is designed to allow users flexibility in retrieving sequences (reads, ORFs, predicted peptides) and search results for focused secondary analyses.

Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.

Construction of DNA fragment libraries for next-generation sequencing can prove challenging, especially for samples with low DNA yield. Protocols devised to circumvent the problems associated with low starting quantities of DNA can result in amplification biases that skew the distribution of genomes in metagenomic data. Moreover, sample throughput can be slow, as current library construction techniques are time-consuming. This study evaluated Nextera, a new transposon-based method that is designed for quick production of DNA fragment libraries from a small quantity of DNA. The sequence read distribution across nine phage genomes in a mock viral assemblage met predictions for six of the least-abundant phages; however, the rank order of the most abundant phages differed slightly from predictions. De novo genome assemblies from Nextera libraries provided long contigs spanning over half of the phage genome; in four cases where full-length genome sequences were available for comparison, consensus sequences were found to match over 99% of the genome with near-perfect identity. Analysis of areas of low and high sequence coverage within phage genomes indicated that GC content may influence coverage of sequences from Nextera libraries. Comparisons of phage genomes prepared using both Nextera and a standard 454 FLX Titanium library preparation protocol suggested that the coverage biases according to GC content observed within the Nextera libraries were largely attributable to bias in the Nextera protocol rather than to the 454 sequencing technology. Nevertheless, given suitable sequence coverage, the Nextera protocol produced high-quality data for genomic studies. For metagenomics analyses, effects of GC amplification bias would need to be considered; however, the library preparation standardization that Nextera provides should benefit comparative metagenomic analyses.

Impacts of poultry house environment on poultry litter bacterial community composition.

Viral and bacterial pathogens are a significant economic concern to the US broiler industry and the ecological epicenter for poultry pathogens is the mixture of bedding material, chicken excrement and feathers that comprises the litter of a poultry house. This study used high-throughput sequencing to assess the richness and diversity of poultry litter bacterial communities, and to look for connections between these communities and the environmental characteristics of a poultry house including its history of gangrenous dermatitis (GD). Cluster analysis of 16S rRNA gene sequences revealed differences in the distribution of bacterial phylotypes between Wet and Dry litter samples and between houses. Wet litter contained greater diversity with 90% of total bacterial abundance occurring within the top 214 OTU clusters. In contrast, only 50 clusters accounted for 90% of Dry litter bacterial abundance. The sixth largest OTU cluster across all samples classified as an Arcobacter sp., an emerging human pathogen, occurring in only the Wet litter samples of a house with a modern evaporative cooling system. Ironically, the primary pathogenic clostridial and staphylococcal species associated with GD were not found in any house; however, there were thirteen 16S rRNA gene phylotypes of mostly gram-positive phyla that were unique to GD-affected houses and primarily occurred in Wet litter samples. Overall, the poultry house environment appeared to substantially impact the composition of litter bacterial communities and may play a key role in the emergence of food-borne pathogens.

Protective effects of angiotensin-converting enzyme inhibitors in high-risk African American men with coronary heart disease.

Angiotensin-converting enzyme (ACE) inhibitors have been extensively used for the treatment of patients with cardiovascular disease, but several concerns have been raised about their efficacy in African American (AA) patients with heart failure, hypertension, and left ventricular hypertrophy. In this study the authors assessed the effect of ACE inhibitors on total and cardiovascular mortality in high-risk AA patients with angiographically proven coronary artery disease (CAD). This was a retrospective analysis of 810 AA men who underwent diagnostic coronary angiography between 1995 and 2003. All patients had demonstrable CAD and had undergone a complete ischemic workup. Follow-up was from 3 to 10 years. ACE inhibitors were administered to 237 patients, while the remaining 537 patients were not taking ACE inhibitors. Patients taking ACE inhibitors had significantly more comorbidities (hypertension, diabetes, left ventricular hypertrophy, heart failure, severe CAD) at baseline, compared with patients not taking ACE inhibitors (P<.05 for all comorbidities). Despite the unfavorable baseline profile, patients taking ACE inhibitors had significantly lower mortality from CAD during follow-up than patients who were not taking ACE inhibitors (P=.006). Stroke mortality rates were similar in both groups. Cox regression analysis showed an 80% higher relative risk in patients not receiving ACE inhibitors. These data indicate a substantial benefit from ACE inhibitor therapy in high-risk AA patients with CAD.