Identification and correction of systematic error in high-throughput sequence data

A feature common to all DNA sequencing technologies is the presence of base-call errors in the sequenced reads. The implications of such errors are application specific, ranging from minor informatics nuisances to major problems affecting biological inferences. Recently developed “next-gen” sequencing technologies have greatly reduced the cost of sequencing, but have been shown to be more error prone than previous technologies. Both position specific (depending on the location in the read) and sequence specific (depending on the sequence in the read) errors have been identified in Illumina and Life Technology sequencing platforms. We describe a new type of _systematic_ error that manifests as statistically unlikely accumulations of errors at specific genome (or transcriptome) locations. We characterize and describe systematic errors using overlapping paired reads form high-coverage data. We show that such errors occur in approximately 1 in 1000 base pairs, and that quality scores at systematic error sites do not account for the extent of errors. We identify motifs that are frequent at systematic error sites, and describe a classifier that distinguishes heterozygous sites from systematic error. Our classifier is designed to accommodate data from experiments in which the allele frequencies at heterozygous sites are not necessarily 0.5 (such as in the case of RNA-Seq). Systematic errors can easily be mistaken for heterozygous sites in individuals, or for SNPs in population analyses. Systematic errors are particularly problematic in low coverage experiments, or in estimates of allele-specific expression from RNA-Seq data. Our characterization of systematic error has allowed us to develop a program, called SysCall, for identifying and correcting such errors. We conclude that correction of systematic errors is important to consider in the design and interpretation of high-throughput sequencing experiments

A Sustained Dietary Change Increases Epigenetic Variation in Isogenic Mice

Epigenetic changes can be induced by adverse environmental exposures, such as nutritional imbalance, but little is known about the nature or extent of these changes. Here we have explored the epigenomic effects of a sustained nutritional change, excess dietary methyl donors, by assessing genomic CpG methylation patterns in isogenic mice exposed for one or six generations. We find stochastic variation in methylation levels at many loci; exposure to methyl donors increases the magnitude of this variation and the number of variable loci. Several gene ontology categories are significantly overrepresented in genes proximal to these methylation-variable loci, suggesting that certain pathways are susceptible to environmental influence on their epigenetic states. Long-term exposure to the diet (six generations) results in a larger number of loci exhibiting epigenetic variability, suggesting that some of the induced changes are heritable. This finding presents the possibility that epigenetic variation within populations can be induced by environmental change, providing a vehicle for disease predisposition and possibly a substrate for natural selection.This work was supported by the Australian Research Council (DP0771859) and the National Health and Medical Research Council (#459412, #635510)

A Generalization of Martin's Axiom

We define the $\aleph_{1.5}$ chain condition. The corresponding forcing axiom is a generalization of Martin's Axiom and implies certain uniform failures of club--guessing on $\omega_1$ that don't seem to have been considered in the literature before.Comment: 36 page

CpG Methylation of a Silent Controlling Element in the Murine Avy Allele Is Incomplete and Unresponsive to Methyl Donor Supplementation

Background: The viable yellow allele of agouti (A vy) is remarkable for its unstable and partially heritable epigenetic state, which produces wide variation in phenotypes of isogenic mice. In the A vy allele an inserted intracisternal A particle (IAP) acts as a controlling element which deregulates expression of agouti by transcription from the LTR of the IAP; the phenotypic state has been linked to CpG methylation of the LTR. Phenotypic variation between A vy mice indicates that the epigenetic state of the IAP is unstable in the germline. Principal Findings: We have made a detailed examination of somatic methylation of the IAP using bisulphite allelic sequencing, and find that the promoter is incompletely methylated even when it is transcriptionally silent. In utero exposure to supplementary methyl donors, which alters the spectrum of A vy phenotypes, does not increase the density of CpG methylation in the silent LTR. Conclusions: Our findings suggest that, contrary to previous supposition, methyl donor supplementation acts through an indirect mechanism to silence A vy. The incomplete cytosine methylation we observe at the somatically silent A vy allele ma

MetMap Enables Genome-Scale Methyltyping for Determining Methylation States in Populations

The ability to assay genome-scale methylation patterns using high-throughput sequencing makes it possible to carry out association studies to determine the relationship between epigenetic variation and phenotype. While bisulfite sequencing can determine a methylome at high resolution, cost inhibits its use in comparative and population studies. MethylSeq, based on sequencing of fragment ends produced by a methylation-sensitive restriction enzyme, is a method for methyltyping (survey of methylation states) and is a site-specific and cost-effective alternative to whole-genome bisulfite sequencing. Despite its advantages, the use of MethylSeq has been restricted by biases in MethylSeq data that complicate the determination of methyltypes. Here we introduce a statistical method, MetMap, that produces corrected site-specific methylation states from MethylSeq experiments and annotates unmethylated islands across the genome. MetMap integrates genome sequence information with experimental data, in a statistically sound and cohesive Bayesian Network. It infers the extent of methylation at individual CGs and across regions, and serves as a framework for comparative methylation analysis within and among species. We validated MetMap's inferences with direct bisulfite sequencing, showing that the methylation status of sites and islands is accurately inferred. We used MetMap to analyze MethylSeq data from four human neutrophil samples, identifying novel, highly unmethylated islands that are invisible to sequence-based annotation strategies. The combination of MethylSeq and MetMap is a powerful and cost-effective tool for determining genome-scale methyltypes suitable for comparative and association studies

Inhibition of Respiration by Nitric Oxide Induces a Mycobacterium tuberculosis Dormancy Program

An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis

Dynamical Masses for Pre-Main Sequence Stars: A Preliminary Physical Orbit for V773 Tau A

We report on interferometric and radial-velocity observations of the double-lined 51-d period binary (A) component of the quadruple pre-main sequence (PMS) system V773 Tau. With these observations we have estimated preliminary visual and physical orbits of the V773 Tau A subsystem. Among other parameters, our orbit model includes an inclination of 66.0 $\pm$ 2.4 deg, and allows us to infer the component dynamical masses and system distance. In particular we find component masses of 1.54 $\pm$ 0.14 and 1.332 $\pm$ 0.097 M_{\sun} for the Aa (primary) and Ab (secondary) components respectively. Our modeling of the subsystem component spectral energy distributions finds temperatures and luminosities consistent with previous studies, and coupled with the component mass estimates allows for comparison with PMS stellar models in the intermediate-mass range. We compare V773 Tau A component properties with several popular solar-composition models for intermediate-mass PMS stars. All models predict masses consistent to within 2-sigma of the dynamically determined values, though some models predict values that are more consistent than others.Comment: ApJ in press; 25 pages, 6 figures; data tables available in journal versio

Proximity to Pollution Sources and Risk of Amphibian Limb Malformation

The cause of limb deformities in wild amphibian populations remains unclear, even though the apparent increase in prevalence of this condition may have implications for human health. Few studies have simultaneously assessed the effect of multiple exposures on the risk of limb deformities. In a cross-sectional survey of 5,264 hylid and ranid metamorphs in 42 Vermont wetlands, we assessed independent risk factors for nontraumatic limb malformation. The rate of nontraumatic limb malformation varied by location from 0 to 10.2%. Analysis of a subsample did not demonstrate any evidence of infection with the parasite Ribeiroia. We used geographic information system (GIS) land-use/land-cover data to validate field observations of land use in the proximity of study wetlands. In a multiple logistic regression model that included land use as well as developmental stage, genus, and water-quality measures, proximity to agricultural land use was associated with an increased risk of limb malformation (odds ratio = 2.26; 95% confidence interval, 1.42–3.58; p < 0.001). The overall discriminant power of the statistical model was high (C = 0.79). These findings from one of the largest systematic surveys to date provide support for the role of chemical toxicants in the development of amphibian limb malformation and demonstrate the value of an epidemiologic approach to this problem

Wedge states in string field theory

The wedge states form an important subalgebra in the string field theory. We review and further investigate their various properties. We find in particular a novel expression for the wedge states, which allows to understand their star products purely algebraically. The method allows also for treating the matter and ghost sectors separately. It turns out, that wedge states with different matter and ghost parts violate the associativity of the algebra. We introduce and study also wedge states with insertions of local operators and show how they are useful for obtaining exact results about convergence of level truncation calculations. These results help to clarify the issue of anomalies related to the identity and some exterior derivations in the string field algebra.Comment: 40 pages, 9 figures, v3: section 3.3 rewritten, few other corrections, set in JHEP styl

Low power radiometric partial discharge sensor using composite transistor-reset integrator

The measurement of partial discharge provides a means of monitoring insulation health in high-voltage equipment. Traditional partial discharge measurements require separate installation for each item of plant to physically connect sensors with specific items. Wireless measurement methods provide an attractive and scalable alternative. Existing wireless monitoring technologies which use time-difference-of-arrival of a partial discharge signal at multiple, spatially separated, sensors place high demands on power consumption and cost due to a requirement for rapid sampling. A recently proposed partial discharge monitoring system using a wireless sensor network and measuring received signal strength only, has potential cost and scalability advantages. An incoherent wireless sensor incorporating a transistor-reset integrator has been developed that reduces the measurement bandwidth of the PD events and alleviates the need for high-speed sampling. It is based on composite amplifier techniques to reduce the power requirements by a factor of approximately four without compromising precision. The accuracy of the proposed sensor is compared to that obtained using a high-speed digital sampling oscilloscope. Received energies were measured over a 10 m distance in 1 m increments and produced an error within 1 dB beyond 4 m and 3.2 dB at shorter distances, resulting in a measurement accuracy within 1 m