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This study proposes an improved semi-analytical approach for contact stiffness modeling of bolted joints in a machine tool system. First, nonlinear contact stress distribution within a single-bolted joint is obtained from the simulation results of finite element analysis software. Second, employing the Hertz contact theory and fractal theory, the contact stiffness model of a single asperity is formulated, affording analytical expressions for normal and tangential contact stiffnesses of a single-bolted joint by integrating multi-asperities in the contact area. Subsequently, considering two test specimens as illustrations, the mode shapes and natural frequencies of the proposed model and modal analysis tests are compared, and the influence of coupling effects between two adjacent bolts is illustrated. The maximum error in the natural frequencies of the proposed approach is

The geomorphological stability and ecological environment of megadeltas worldwide are of vital importance for their sustainable development. Deltaic hydro-morphodynamics is extremely sensitive to high riverine flow due to reduced sediment supply. However, the morphological evolution and response of deltas under high riverine flow have remained inadequately quantified. As one of the typical megadeltas, the Yellow River Delta (YRD), is becoming increasingly sensitive to environmental changes and intensified human interventions. In this study, a numerical model and field data were used to investigate the hydrodynamic changes and morphodynamic evolution induced by extreme river discharge in the YRD. The numerical experiments with different runoff scenarios reveal that high-energy riverine floods can cause significant hydrodynamic changes in bed shear stresses, water levels, and flow velocities, particularly in the abandoned river mouth. Moreover, it enhances the ebb-dominated tidal asymmetry, which considerably intensifies fluvial sediment resuspension and transport processes. The results also show high-energy riverine floods in the flood seasons trigger severe erosion in the Yellow River submerged delta, with a net erosion volume reaching −0.07 × 108 m3/year. The hydrodynamic increment in the abandoned river mouth is more significant, and therefore, severe erosion occurs, with the maximum erosion thickness reaching 7 m. These findings highlight the role of high riverine floods on the hydro-sediment dynamics of large river deltas under a sediment starvation condition.

Nonlinear unloading plays an important role in predicting springback during plastic forming process. To improve the accuracy of springback prediction which could provide a guide for precision forming, uniaxial tensile tests and uniaxial loading–unloading–loading tensile tests on SUS304 stainless steel were carried out. The flow stress mathematical model and chord modulus mathematical model were calibrated according to the test results. A constant elastic modulus three-point bending finite element model (E0FEMB) and a constant elastic modulus roll forming finite element model (E0FEMR) were established in MSC.MARC. The chord modulus was output by the PLOTV subroutine to determine the mean modulus of different regions, and the mean modulus three-point bending finite element model (E¯cFEMB) and the mean modulus roll forming finite element model (E¯cFEMR) were defined. The constant modulus finite element model (E0FEM) simulation results and the mean modulus finite element model (E¯cFEM) simulation results were compared with the three-point bending tests and roll forming tests test results. The difference between the simulation results and the test results was small, indicating that the mean modulus was feasible to predict the springback, which verified the suitability of the E¯cFEM.

Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

The dimming control system with high efficiency and energy saving, simple and convenient operation is of great significance to daily life and industrial production. This paper we uses STM32 as the control core, designs a dimming control system based on PWM dimming mode and Bluetooth to send control commands. The experimental results show that the system can realize the smooth adjustment of the brightness of the four LEDs remotely

River dynamics and sediment budget play a crucial role in shaping geomorphic variability of river channels and deltaic environments. Basin-scale human activities, including dam construction, induce alterations in river flow and sediment dynamics in the downstream channels and to the delta, and quantification of sediment source shift along downstream fluvial-deltaic systems is often uncertain. This study analyzed the river regime changes and sediment dynamics of a typical sediment-laden fluvial-deltaic system—the lower Yellow River (LYR) and the Yellow River Delta (YRD) —to assess the integrated effects of dam impoundment and dam-based river regulation schemes on downstream hydrogeomorphic transition processes. The Xiaolangdi (XLD) Reservoir, which was completed in 2000 with a total storage of 12.7 km3, is the final reservoir located in the middle Yellow River and plays an important role in flood control and energy supply. Following the full operation of XLD Reservoir, the relationship between water and sediment in the LYR became more balanced, with a drastic decline of sediment input and seasonal migrations of floodwaters. The interannual variability of water levels at downstream hydrological stations indicated a geomorphic transition in the LYR from net deposition to erosion state. The building of the XLD Reservoir caused a downstream shift of river-originated sediment source and 48% of the total sediment delivered to the YRD was derived from the LYR. However, the reduced sediment delivery since 2000 has still triggered net land loss regarding the YRD system, with a strong spatial variability which is dominated by the reduced accretion at the active delta front and erosion at the abandoned river mouth and coastal engineering zone. Compared with other environmental factors, the construction of upstream dams contributed the most to the decline of downstream sediment delivery over the past decades. The challenge for sustainable sediment management is the gradual decline of scouring efficiency as the riverbed sediment is coarsening. Our study suggests that future river regulation strategies should consider the geomorphic sustainability of both the LYR and the YRD system.

Suspended particulate matter (SPM) is the primary component of coastal waters. The spatiotemporal dynamics of SPM has an important influence on water bio-optical properties, pollutant diffusion, landform evolution, and carbon, oxygen, and nutrient cycles. This study presents the spatial distribution of monthly average SPM concentrations from 2017 to 2020 in the abandoned Diaokouhe and Shenxiangou distributary mouths of the Yellow River Delta (YRD). The SPM concentrations were derived based on 161 Landsat-8 OLI and Sentinel-2A/B MSI satellite images. Accuracy assessment using the synchronous in-situ measurements (R2 = 0.91, root mean square error (RMSE) = 38.79 mg/L, and relative percentage difference (RPD) = 23.45%) and cross comparison between SPM concentrations derived from the two sensors (R2 = 0.97, RPD = 8.77%) showed convincing performances. Our results show that the monthly average SPM is characterized by significant seasonality, with a higher concentration and wider range in dry seasons compared to that in wet seasons. The spatial distributions of SPM are closely associated with coastal water depths. The SPM concentration in the Shenxiangou mouth is always lower than that of the shallower Diaokouhe mouth. The long-term seabed erosion provides a continuous supply of sediment. Wind-wave forces explain the seasonal variations of SPM by inducing the resuspension of bottom sediments. The residual currents control the transportation of SPM to the offshore and to the east. The artificial groins located between the two river mouths show obvious sediment trapping effects. Moreover, the interception capability of impermeable groins is considerably better than that of permeable groins.

Sustainable development of megadeltas worldwide has recently been the focus of international socioeconomic and environmental concerns. Long-term geomorphic sustainability of deltas is crucial for sustaining coastal resources and marine ecosystems. The modern Yellow River Delta (YRD), which has been developed since 1855, is increasingly facing erosion threats under natural and human-induced changes. Utilizing long-term hydrological data, remote-sensing satellite images and bathymetric survey data, the transition processes of the modern YRD (including both the subaerial and subaqueous delta) during 1855–2015 from the perspective of morphodynamics are examined. The results show that over the past 160 years, the evolution of the Yellow River subaerial delta is under rapid accretion before 2000, and transitioned to slight erosion state after 2000, which is believed to be associated with the reduced sediment supply. Correspondingly, the Yellow River subaqueous delta is also believed to transition from the accretion into net erosion since 2000, with a net volume change rate of −1.5 × 108 m3/yr. As the primary source for deltaic deposits, at least 2.6 × 108 t/yr of sediment supply is required to sustain the erosion-accretion balance of both the subaerial and subaqueous delta. Compared with the previous research, we present that the 160-year morphological evolution of the modern deltaic system is strongly influenced by the reduction of sediment input. Within this time frame the changes in the partition of sediment load among frequent migrations of the deltaic channel could have a direct impact on variations of the long-term erosion-deposition patterns of the modern YRD. Extreme events of storm surges and frequent strong winds caused significant short-term fluctuations in the erosion/deposition of the subaqueous delta that would affect long-term changes. The insights gained from this study may help to set scientific guidelines for the sustainable development of other river deltas worldwide that are subject to climate change and human interventions.

Background The human genome contains variants ranging in size from small single nucleotide polymorphisms (SNPs) to large structural variants (SVs). High-quality benchmark small variant calls for the pilot National Institute of Standards and Technology (NIST) Reference Material (NA12878) have been developed by the Genome in a Bottle Consortium, but no similar high-quality benchmark SV calls exist for this genome. Since SV callers output highly discordant results, we developed methods to combine multiple forms of evidence from multiple sequencing technologies to classify candidate SVs into likely true or false positives. Our method (svclassify) calculates annotations from one or more aligned bam files from many high-throughput sequencing technologies, and then builds a one-class model using these annotations to classify candidate SVs as likely true or false positives. Results We first used pedigree analysis to develop a set of high-confidence breakpoint-resolved large deletions. We then used svclassify to cluster and classify these deletions as well as a set of high-confidence deletions from the 1000 Genomes Project and a set of breakpoint-resolved complex insertions from Spiral Genetics. We find that likely SVs cluster separately from likely non-SVs based on our annotations, and that the SVs cluster into different types of deletions. We then developed a supervised one-class classification method that uses a training set of random non-SV regions to determine whether candidate SVs have abnormal annotations different from most of the genome. To test this classification method, we use our pedigree-based breakpoint-resolved SVs, SVs validated by the 1000 Genomes Project, and assembly-based breakpoint-resolved insertions, along with semi-automated visualization using svviz. Conclusions We find that candidate SVs with high scores from multiple technologies have high concordance with PCR validation and an orthogonal consensus method MetaSV (99.7 % concordant), and candidate SVs with low scores are questionable. We distribute a set of 2676 high-confidence deletions and 68 high-confidence insertions with high svclassify scores from these call sets for benchmarking SV callers. We expect these methods to be particularly useful for establishing high-confidence SV calls for benchmark samples that have been characterized by multiple technologies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2366-2) contains supplementary material, which is available to authorized users.

Caffeine is the most widely consumed psychoactive substance and has complex pharmacological actions in brain. In this study, we employed a novel drug target validation strategy to uncover the multiple molecular targets of caffeine using combined A2Areceptor (A2AR) knockouts (KO) and microarray profiling. Caffeine (10 mg/kg) elicited a distinct profile of striatal gene expression in WT mice compared with that by A2AR gene deletion or by administering caffeine into A2AR KO mice. Thus, A2ARs are required but not sufficient to elicit the striatal gene expression by caffeine (10 mg/kg). Caffeine (50 mg/kg) induced complex expression patterns with three distinct sets of striatal genes: 1) one subset overlapped with those elicited by genetic deletion of A2ARs; 2) the second subset elicited by caffeine in WT as well as A2AR KO mice; and 3) the third subset elicited by caffeine only in A2AR KO mice. Furthermore, striatal gene sets elicited by the phosphodiesterase (PDE) inhibitor rolipram and the GABAAreceptor antagonist bicucullin, overlapped with the distinct subsets of striatal genes elicited by caffeine (50 mg/kg) administered to A2AR KO mice. Finally, Gene Set Enrichment Analysis reveals that adipocyte differentiation/insulin signaling is highly enriched in the striatal gene sets elicited by both low and high doses of caffeine. The identification of these distinct striatal gene populations and their corresponding multiple molecular targets, including A2AR, non-A2AR (possibly A1Rs and pathways associated with PDE and GABAAR) and their interactions, and the cellular pathways affected by low and high doses of caffeine, provides molecular insights into the acute pharmacological effects of caffeine in the brain.

A set of 723 high-quality human core promoter sequences were compiled and analyzed for overrepresented motifs. Beside the two well-characterized core promoter motifs (TATA and Inr), several known motifs (YY1, Sp1, NRF-1, NRF-2, CAAT, and CREB) and one potentially new motif (motif8) were found. Interestingly, YY1 and motif8 mostly reside immediately downstream from the TSS. In particular, the YY1 motif occurs primarily in genes with 5′-UTRs shorter than 40 base pairs (bp) and its locations coincide with the translation start site. We verified that the YY1 motif is bound by YY1 in vitro. We then performed detailed analysis on YY1 chromatin immunoprecipitation data with a whole-genome human promoter microarray (ChIP-chip) and revealed that the thus identified promoters in HeLa cells were highly enriched with the YY1 motif. Moreover, the motif overlapped with the translation start sites on the plus strand of a group of genes, many with short 5′-UTRs, and with the transcription start sites on the minus strand of another distinct group of genes; together, the two groups of genes accounted for the majority of the YY1-bound promoters in the ChIP-chip data. Furthermore, the first group of genes was highly enriched in the functional categories of ribosomal proteins and nuclear-encoded mitochondria proteins. We suggest that the YY1 motif plays a dual role in both transcription and translation initiation of these genes. We also discuss the evolutionary advantages of housing a transcriptional element inside the transcript in terms of the migration of these genes in the human genome.

Eukaryotic gene transcription is accompanied by acetylation and methylation of nucleosomes near promoters, but the locations and roles of histone modifications elsewhere in the genome remain unclear. We determined the chromatin modification states in high resolution along 30 Mb of the human genome and found that active promoters are marked by trimethylation of Lys4 of histone H3 (H3K4), whereas enhancers are marked by monomethylation, but not trimethylation, of H3K4. We developed computational algorithms using these distinct chromatin signatures to identify new regulatory elements, predicting over 200 promoters and 400 enhancers within the 30-Mb region. This approach accurately predicted the location and function of independently identified regulatory elements with high sensitivity and specificity and uncovered a novel functional enhancer for the carnitine transporter SLC22A5 (OCTN2). Our results give insight into the connections between chromatin modifications and transcriptional regulatory activity and provide a new tool for the functional annotation of the human genome.

Since more and more advertisements swarm into radios, it is necessary to establish an audio advertising dataset which could be used to analyze and classify the advertisement. A method of how to establish a complete audio advertising dataset is presented in this paper. The dataset is divided into four different kinds of advertisements. Each advertisement’s sample is given in *.wav file format, and annotated with a txt file which contains its file name, sampling frequency, channel number, broadcasting time and its class. The classifying rationality of the advertisements in this dataset is proved by clustering the different advertisements based on Principal Component Analysis (PCA). The experimental results show that this audio advertisement dataset offers a reliable set of samples for correlative audio advertisement experimental studies.

Considering the huge global influence of Ebola, its an urgent task to think out an approach to eradicate its spread. In this paper, we build a Prediction-Prevention mathematic model which succeeds in optimizing the eradication of Ebola. We also call it the P&P model. Firstly, we establish the RISK (Recovered-Infected-Susceptible-Isolated) epidemic model, which is the modified version of classic SIR (Susceptible-Infected-Recovered) epidemic model. Secondly, we establish the Delivery model to better describe the delivery mechanism. The Delivery model is composed of three sub-models: The production model, the transportation model and the distribution model. Finally, the relationship between the RISK model and the delivery model are established. The results show that our model has good robustness and thus more reliable.

The protooncogene MYC encodes the c-Myc transcription factor that regulates cell growth, cell proliferation, cell cycle, and apoptosis. Although deregulation of MYC contributes to tumorigenesis, it is still unclear what direct Myc-induced transcriptomes promote cell transformation. Here we provide a snapshot of genome-wide, unbiased characterization of direct Myc binding targets in a model of human B lymphoid tumor using ChIP coupled with pair-end ditag sequencing analysis (ChIP-PET). Myc potentially occupies >4,000 genomic loci with the majority near proximal promoter regions associated frequently with CpG islands. Using gene expression profiles with ChIP-PET, we identified 668 direct Myc-regulated gene targets, including 48 transcription factors, indicating that Myc is a central transcriptional hub in growth and proliferation control. This first global genomic view of Myc binding sites yields insights of transcriptional circuitries and cis regulatory modules involving Myc and provides a substantial framework for our understanding of mechanisms of Myc-induced tumorigenesis.

The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (∼1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.

Detecting overrepresented known transcription factor binding motifs in a set of promoter sequences of co-regulated genes has become an important approach to deciphering transcriptional regulatory mechanisms. In this paper, we present an interactive web server, MotifViz, for three motif discovery programs, Clover, Rover and Motifish, covering most available flavors of algorithms for achieving this goal. For comparison, we have also implemented the simple motif-matching program Possum. MotifViz provides uniform and intuitive input and output formats for all four programs. It can be accessed at http://biowulf.bu.edu/MotifViz.

Transcriptional regulation is one of the most basic regulatory mechanisms in the cell. The accumulation of multiple metazoan genome sequences and the advent of high-throughput experimental techniques have motivated the development of a large number of bioinformatics methods for the detection of regulatory motifs. The regulatory process is extremely complex and individual computational algorithms typically have very limited success in genome-scale studies. Here, we argue the importance of integrating multiple computational algorithms and present an infrastructure that integrates eight web services covering key areas of transcriptional regulation. We have adopted the client-side integration technology and built a consistent input and output environment with a versatile visualization tool named SeqVISTA. The infrastructure will allow for easy integration of gene regulation analysis software that is scattered over the Internet. It will also enable bench biologists to perform an arsenal of analysis using cutting-edge methods in a familiar environment and bioinformatics researchers to focus on developing new algorithms without the need to invest substantial effort on complex pre- or post-processors. SeqVISTA is freely available to academic users and can be launched online at http://zlab.bu.edu/SeqVISTA/web.jnlp, provided that Java Web Start has been installed. In addition, a stand-alone version of the program can be downloaded and run locally. It can be obtained at http://zlab.bu.edu/SeqVISTA.

The interaction of proteins with DNA recognition motifs regulates a number of fundamental biological processes, including transcription. To understand these processes, we need to know which motifs are present in a sequence and which factors bind to them. We describe a method to screen a set of DNA sequences against a precompiled library of motifs, and assess which, if any, of the motifs are statistically over- or under-represented in the sequences. Over-represented motifs are good candidates for playing a functional role in the sequences, while under-representation hints that if the motif were present, it would have a harmful dysregulatory effect. We apply our method (implemented as a computer program called Clover) to dopamine-responsive promoters, sequences flanking binding sites for the transcription factor LSF, sequences that direct transcription in muscle and liver, and Drosophila segmentation enhancers. In each case Clover successfully detects motifs known to function in the sequences, and intriguing and testable hypotheses are made concerning additional motifs. Clover compares favorably with an ab initio motif discovery algorithm based on sequence alignment, when the motif library includes only a homolog of the factor that actually regulates the sequences. It also demonstrates superior performance over two contingency table based over-representation methods. In conclusion, Clover has the potential to greatly accelerate characterization of signals that regulate transcription.

Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli , Enterobacter aerogenes , and Pseudomonas putida. Micromonas pusilla , an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida , the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.

We have implemented aggregation and correlation toolbox (ACT), an efficient, multifaceted toolbox for analyzing continuous signal and discrete region tracks from high-throughput genomic experiments, such as RNA-seq or ChIP-chip signal profiles from the ENCODE and modENCODE projects, or lists of single nucleotide polymorphisms from the 1000 genomes project. It is able to generate aggregate profiles of a given track around a set of specified anchor points, such as transcription start sites. It is also able to correlate related tracks and analyze them for saturation–i.e. how much of a certain feature is covered with each new succeeding experiment. The ACT site contains downloadable code in a variety of formats, interactive web servers (for use on small quantities of data), example datasets, documentation and a gallery of outputs. Here, we explain the components of the toolbox in more detail and apply them in various contexts. Availability: ACT is available at http://act.gersteinlab.org Contact: gro.balnietsreg@ip

Retrieving the wanted contents within large amount of video quickly is one of the key technologies of advertisement recognition. This paper analyzed the principle of AdaBoost algorithm and SURF matching algorithm, and designed a system of target face retrieving. The Adaboost algorithm was used to detect the faces from video key flames, and SURF matching algorithm was used to match the targeted face in a video. This system especially solved the matching problem for the changed targeted face brought by the different angle of view and scale in the video. This work was programmed by C# language in the environment of Visual Studio 2010 and EmguCV. The experimental results showed that the system could retrieve the appearing time of the target face accurately, and it was easy to be operated and was with a certain robustness and practicability.

Comprehensive identification of the acquired mutations that cause common cancers will require genomic analyses of large sets of tumor samples. Typically, the tissue material available from tumor specimens is limited, which creates a demand for accurate template amplification. We therefore evaluated whether phi29-mediated whole genome amplification introduces false positive structural mutations by massive mate-pair sequencing of a normal human genome before and after such amplification. Multiple displacement amplification led to a decrease in clone coverage and an increase by two orders of magnitude in the prevalence of inversions, but did not increase the prevalence of translocations. While multiple strand displacement amplification may find uses in translocation analyses, it is likely that alternative amplification strategies need to be developed to meet the demands of cancer genomics. 462IGP

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

Abnormalities of genomic methylation patterns are lethal or cause disease, but the cues that normally designate CpG dinucleotides for methylation are poorly understood. We have developed a new method of methylation profiling that has single-CpG resolution and can address the methylation status of repeated sequences. We have used this method to determine the methylation status of >275 million CpG sites in human and mouse DNA from breast and brain tissues. Methylation density at most sequences was found to increase linearly with CpG density and to fall sharply at very high CpG densities, but transposons remained densely methylated even at higher CpG densities. The presence of histone H2A.Z and histone H3 di- or trimethylated at lysine 4 correlated strongly with unmethylated DNA and occurred primarily at promoter regions. We conclude that methylation is the default state of most CpG dinucleotides in the mammalian genome and that a combination of local dinucleotide frequencies, the interaction of repeated sequences, and the presence or absence of histone variants or modifications shields a population of CpG sites (most of which are in and around promoters) from DNA methyltransferases that lack intrinsic sequence specificity.

Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD™ System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD™ bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding ∼18× haploid coverage of aligned sequence and close to 300× clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.

The most widely used method for detecting genome-wide protein–DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and “spike-ins” comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols, and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.

The comprehensive inventory of functional elements in 44 human genomic regions carried out by the ENCODE Project Consortium enables for the first time a global analysis of the genomic distribution of transcriptional regulatory elements. In this study we developed an intuitive and yet powerful approach to analyze the distribution of regulatory elements found in many different ChIP–chip experiments on a 10∼100-kb scale. First, we focus on the overall chromosomal distribution of regulatory elements in the ENCODE regions and show that it is highly nonuniform. We demonstrate, in fact, that regulatory elements are associated with the location of known genes. Further examination on a local, single-gene scale shows an enrichment of regulatory elements near both transcription start and end sites. Our results indicate that overall these elements are clustered into regulatory rich “islands” and poor “deserts.” Next, we examine how consistent the nonuniform distribution is between different transcription factors. We perform on all the factors a multivariate analysis in the framework of a biplot, which enhances biological signals in the experiments. This groups transcription factors into sequence-specific and sequence-nonspecific clusters. Moreover, with experimental variation carefully controlled, detailed correlations show that the distribution of sites was generally reproducible for a specific factor between different laboratories and microarray platforms. Data sets associated with histone modifications have particularly strong correlations. Finally, we show how the correlations between factors change when only regulatory elements far from the transcription start sites are considered.

Bidirectional promoters have received considerable attention because of their ability to regulate two downstream genes (divergent genes). They are also highly abundant, directing the transcription of ∼11% of genes in the human genome. We categorized the presence of DNA sequence motifs, binding of transcription factors, and modified histones as overrepresented, shared, or underrepresented in bidirectional promoters with respect to unidirectional promoters. We found that a small set of motifs, including GABPA, MYC, E2F1, E2F4, NRF-1, CCAAT, YY1, and ACTACAnnTCC are overrepresented in bidirectional promoters, while the majority (73%) of known vertebrate motifs are underrepresented. We performed chromatin-immunoprecipitation (ChIP), followed by quantitative PCR for GABPA, on 118 regions in the human genome and showed that it binds to bidirectional promoters more frequently than unidirectional promoters, and its position-specific scoring matrix is highly predictive of binding. Signatures of active transcription, such as occupancy of RNA polymerase II and the modified histones H3K4me2, H3K4me3, and H3ac, are overrepresented in regions around bidirectional promoters, suggesting that a higher fraction of divergent genes are transcribed in a given cell than the fraction of other genes. Accordingly, analysis of whole-genome microarray data indicates that 68% of divergent genes are transcribed compared with 44% of all human genes. By combining the analysis of publicly available ENCODE data and a detailed study of GABPA, we survey bidirectional promoters with breadth and depth, leading to biological insights concerning their motif composition and bidirectional regulatory mode.