A Fourier characteristic of coding sequences: origins and a non-Fourier approximation.

The 3-base periodicity, identified as a pronounced peak at the frequency N/3 (N is the length of the DNA sequence) of the Fourier power spectrum of protein coding regions, is used as a marker in gene-finding algorithms to distinguish protein coding regions (exons) and noncoding regions (introns) of genomes. In this paper, we reveal the explanation of this phenomenon which results from a nonuniform distribution of nucleotides in the three coding positions. There is a linear correlation between the nucleotide distributions in the three codon positions and the power spectrum at the frequency N/3. Furthermore, this study indicates the relationship between the length of a DNA sequence and the variance of nucleotide distributions and the average Fourier power spectrum, which is the noise signal in gene-finding methods. The results presented in this paper provide an efficient way to compute the Fourier power spectrum at N/3 and the noise signal in gene-finding methods by calculating the nucleotide distributions in the three codon positions.     

Action at Hooked or Twisted-Hooked DNA Juxtapositions Rationalizes Unlinking Preference of Type-2 Topoisomerases

The mathematical basis of the hypothesis that type-2 topoisomerases recognize and act at specific DNA juxtapositions has been investigated by coarse-grained lattice polymer models, showing that selective segment passages at hooked juxtapositions can result in dramatic reductions in catenane and knot populations. The lattice modeling approach is here extended to account for the narrowing of variance of linking number (Lk) of DNA circles by type-2 topoisomerases. In general, the steady-state variance of Lk resulting from selective segment passages at a specific juxtaposition geometry j is inversely proportional to the average linking number, 〈Lk〉_j, of circles with the given juxtaposition. Based on this formulation, we demonstrate that selective segment passages at hooked juxtapositions reduce the variance of Lk. The dependence of this effect on model DNA circle size is remarkably similar to that observed experimentally for type-2 topoisomerases, which appear to be less capable in narrowing Lk variance for small DNA circles than for larger DNA circles. This behavior is rationalized by a substantial cancellation of writhe in small circles with hook-like juxtapositions. During our simulations, we uncovered a twisted variation of the hooked juxtaposition that has an even more dramatic effect on Lk variance narrowing than the hooked juxtaposition. For an extended set of juxtapositions, we detected a significant correlation between the Lk narrowing potential and the logarithmic decatenating and unknotting potentials for a given juxtaposition, a trend reminiscent of scaling relations observed with experimental measurements on type-2 topoisomerases from a variety of organisms. The consistent agreement between theory and experiment argues for type-2 topoisomerase action at hooked or twisted-hooked DNA juxtapositions.     

DNA binding spectrum of the carcinogen N-acetoxy-N-2-acetylaminofluorene significantly differs from the mutation spectrum

The 3′ → 5′ exonuclease activity of bacteriophage T4 DNA polymerase is found to be blocked in the vicinity of the N-2-acetylaminofluorene (-AAF) adducts to DNA. This observation allowed us to determine the binding spectrum of the -AAF adducts along a given DNA sequence. The mutation spectrum in a forward mutation assay within this same sequence has been established. Comparison between the -AAF binding spectrum and the mutation spectrum shows that there is no direct correlation.     

X-inactivation informs variance-based testing for X-linked association of a quantitative trait

Background

The X chromosome plays an important role in human diseases and traits. However, few X-linked associations have been reported in genome-wide association studies, partly due to analytical complications and low statistical power.

Results

In this study, we propose tests of X-linked association that capitalize on variance heterogeneity caused by various factors, predominantly the process of X-inactivation. In the presence of X-inactivation, the expression of one copy of the chromosome is randomly silenced. Due to the consequent elevated randomness of expressed variants, females that are heterozygotes for a quantitative trait locus might exhibit higher phenotypic variance for that trait. We propose three tests that build on this phenomenon: 1) A test for inflated variance in heterozygous females; 2) A weighted association test; and 3) A combined test. Test 1 captures the novel signal proposed herein by directly testing for higher phenotypic variance of heterozygous than homozygous females. As a test of variance it is generally less powerful than standard tests of association that consider means, which is supported by extensive simulations. Test 2 is similar to a standard association test in considering the phenotypic mean, but differs by accounting for (rather than testing) the variance heterogeneity. As expected in light of X-inactivation, this test is slightly more powerful than a standard association test. Finally, test 3 further improves power by combining the results of the first two tests. We applied the these tests to the ARIC cohort data and identified a novel X-linked association near gene AFF2 with blood pressure, which was not significant based on standard association testing of mean blood pressure.

Conclusions

Variance-based tests examine overdispersion, thereby providing a complementary type of signal to a standard association test. Our results point to the potential to improve power of detecting X-linked associations in the presence of variance heterogeneity.     

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR

Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair. In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy. We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer. This method is used for rapid analysis of mutational spectra generated in nuclease-defective strains of Saccharomyces cerevisiae to elucidate the role of RNase H(35) in RNA primer removal during DNA replication and in mutation avoidance. Strains deficient in RNH35 displayed a distinct spontaneous mutation spectrum of deletions characterized by a unique 4 bp deletion in a lys2-Bgl allele. This was in sharp contrast to strains deficient in rad27 that displayed duplication mutations. Further analysis of mutations in a rnh35/rad27 double mutant revealed a mixed spectrum. These results indicate that RNase H(35) may participate in a redundant pathway in Okazaki fragment processing and that mutational spectra caused by protein deficiencies may be more intermediate-specific than pathway-specific.     

Genetic and Epigenetic Somatic Alterations in Head and Neck Squamous Cell Carcinomas Are Globally Coordinated but Not Locally Targeted

Background

Solid tumors, including head and neck squamous cell carcinomas (HNSCC), arise as a result of genetic and epigenetic alterations in a sustained stress environment. Little work has been done that simultaneously examines the spectrum of both types of changes in human tumors on a genome-wide scale and results so far have been limited and mixed. Since it has been hypothesized that epigenetic alterations may act by providing the second carcinogenic hit in gene silencing, we sought to identify genome-wide DNA copy number alterations and CpG dinucleotide methylation events and examine the global/local relationships between these types of alterations in HNSCC.

Methodology/Principal Findings

We have extended a prior analysis of 1,413 cancer-associated loci for epigenetic changes in HNSCC by integrating DNA copy number alterations, measured at 500,000 polymorphic loci, in a case series of 19 primary HNSCC tumors. We have previously demonstrated that local copy number does not bias methylation measurements in this array platform. Importantly, we found that the global pattern of copy number alterations in these tumors was significantly associated with tumor methylation profiles (p<0.002). However at the local level, gene promoter regions did not exhibit a correlation between copy number and methylation (lowest q = 0.3), and the spectrum of genes affected by each type of alteration was unique.

Conclusion/Significance

This work, using a novel and robust statistical approach demonstrates that, although a “second hit” mechanism is not likely the predominant mode of action for epigenetic dysregulation in cancer, the patterns of methylation events are associated with the patterns of allele loss. Our work further highlights the utility of integrative genomics approaches in exploring the driving somatic alterations in solid tumors.     

Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10⁻² pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.     

Estimating the Growth Rate of Slowly Growing Marine Bacteria from RNA Content

In past studies of enteric bacteria such as Escherichia coli, various measures of cellular RNA content have been shown to be strongly correlated with growth rate. We examined this correlation for four marine bacterial isolates. Isolates were grown in chemostats at four or five dilution rates, yielding growth rates that spanned the range typically determined for marine bacterial communities in nature (μ = 0.01 to 0.25 h^-1). All measures of RNA content (RNA cell^-1, RNA:biovolume ratio, RNA:DNA ratio, RNA:DNA:biovolume ratio) were significantly different among isolates. Normalizing RNA content to cell volume substantially reduced, but did not eliminate, these differences. On average, the correlation between μ and the RNA:DNA ratio accounted for 94% of variance when isolates were considered individually. For data pooled across isolates (analogous to an average measurement for a community), the ratio of RNA:DNA μm^-3 (cell volume) accounted for nearly half of variance in μ (r² = 0.47). The maximum RNA:DNA ratio for each isolate was extrapolated from regressions. The regression of (RNA:DNA)/(RNA:DNA)_max on μ was highly significant (r² = 0.76 for data pooled across four isolates) and virtually identical for three of the four isolates, perhaps reflecting an underlying common relationship between RNA content and growth rate. The dissimilar isolate was the only one derived from sediment. Cellular RNA content is likely to be a useful predictor of growth rate for slowly growing marine bacteria but in practice may be most successful when applied at the level of individual species.     

Joint QTL linkage mapping for multiple-cross mating design sharing one common parent

Background

Nested association mapping (NAM) is a novel genetic mating design that combines the advantages of linkage analysis and association mapping. This design provides opportunities to study the inheritance of complex traits, but also requires more advanced statistical methods. In this paper, we present the detailed algorithm of a QTL linkage mapping method suitable for genetic populations derived from NAM designs. This method is called joint inclusive composite interval mapping (JICIM). Simulations were designed on the detected QTL in a maize NAM population and an Arabidopsis NAM population so as to evaluate the efficiency of the NAM design and the JICIM method.

Principal Findings

Fifty-two QTL were identified in the maize population, explaining 89% of the phenotypic variance of days to silking, and nine QTL were identified in the Arabidopsis population, explaining 83% of the phenotypic variance of flowering time. Simulations indicated that the detection power of these identified QTL was consistently high, especially for large-effect QTL. For rare QTL having significant effects in only one family, the power of correct detection within the 5 cM support interval was around 80% for 1-day effect QTL in the maize population, and for 3-day effect QTL in the Arabidopsis population. For smaller-effect QTL, the power diminished, e.g., it was around 50% for maize QTL with an effect of 0.5 day. When QTL were linked at a distance of 5 cM, the likelihood of mapping them as two distinct QTL was about 70% in the maize population. When the linkage distance was 1 cM, they were more likely mapped as one single QTL at an intermediary position.

Conclusions

Because it takes advantage of the large genetic variation among parental lines and the large population size, NAM is a powerful multiple-cross design for complex trait dissection. JICIM is an efficient and specialty method for the joint QTL linkage mapping of genetic populations derived from the NAM design.     

Further insights of the variance component method for detecting QTL in livestock and aquacultural species: relaxing the assumption of additive effects

Complex traits may show some degree of dominance at the gene level that may influence the statistical power of simple models, i.e. assuming only additive effects to detect quantitative trait loci (QTL) using the variance component method. Little has been published on this topic even in species where relatively large family sizes can be obtained, such as poultry, pigs, and aquacultural species. This is important, when the idea is to select regions likely to be harbouring dominant QTL or in marker assisted selection. In this work, we investigated the empirical power and accuracy to both detect and localise dominant QTL with or without incorporating dominance effects explicitly in the model of analysis. For this purpose, populations with variable family sizes and constant population size and different values for dominance variance were simulated. The results show that when using only additive effects there was little loss in power to detect QTL and estimates of position, using or not using dominance, were empirically unbiased. Further, there was little gain in accuracy of positioning the QTL with most scenarios except when simulating an overdominant QTL.     

Power spectra of extinction in the fossil record

Recent Fourier analyses of fossil extinction data have indicated that the power spectrum of extinction during the Phanerozoic may take the form of 1/f noise, a result which, it has been suggested, could be indicative of the presence of `critical dynamics'' in the processes giving rise to extinction. In this paper we examine extinction power spectra in some detail, using family-level data from two widely available compilations. We find that although the average form of the power spectrum roughly obeys the 1/f law, the spectrum can be represented more accurately by dividing it into two regimes: a low-frequency one which is well fit by an exponential, and a high-frequency one in which it follows a power law with a 1/f² form. We give explanations for the occurrence of each of these behaviours and for the position of the crossover between them.     

Assignment of SNP allelic configuration in polyploids using competitive allele-specific PCR: application to citrus triploid progeny

Background

Polyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids.

Scope

This work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers.

Conclusions

Fifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan⁻¹ (y/x) and y′ = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis.     

Models of Postural Control: Shared Variance in Joint and COM Motions

This paper investigated the organization of the postural control system in human upright stance. To this aim the shared variance between joint and 3D total body center of mass (COM) motions was analyzed using multivariate canonical correlation analysis (CCA). The CCA was performed as a function of established models of postural control that varied in their joint degrees of freedom (DOF), namely, an inverted pendulum ankle model (2DOF), ankle-hip model (4DOF), ankle-knee-hip model (5DOF), and ankle-knee-hip-neck model (7DOF). Healthy young adults performed various postural tasks (two-leg and one-leg quiet stances, voluntary AP and ML sway) on a foam and rigid surface of support. Based on CCA model selection procedures, the amount of shared variance between joint and 3D COM motions and the cross-loading patterns we provide direct evidence of the contribution of multi-DOF postural control mechanisms to human balance. The direct model fitting of CCA showed that incrementing the DOFs in the model through to 7DOF was associated with progressively enhanced shared variance with COM motion. In the 7DOF model, the first canonical function revealed more active involvement of all joints during more challenging one leg stances and dynamic posture tasks. Furthermore, the shared variance was enhanced during the dynamic posture conditions, consistent with a reduction of dimension. This set of outcomes shows directly the degeneracy of multivariate joint regulation in postural control that is influenced by stance and surface of support conditions.     

Structural analysis of hyperperiodic DNA from Caenorhabditis elegans

Several bioinformatics studies have identified an unexpected but remarkably prevalent ~10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties.     

A genome-wide association study of mare fertility in the Pura Raza Español horse

Despite the economic importance of fertility for the horse industry, few efforts have been made to achieve a better understanding of the genetic mechanisms underlying its control. This is probably due to the difficulty of obtaining reliable phenotypes and the complexity of modelling the environmental and management factors. This work is novel in that we propose to use reproductive efficiency (RE) as an indicator of mare fertility. To achieve this, we performed a genome-wide association study in the Pura Raza Español horse aimed at identifying genomic variants, regions, and candidate genes associated with fertility in mares. The dataset included 819 animals genotyped with the Affymetrix Axiom? Equine 670 K single-nucleotide polymorphisms (SNPs) Genotyping Array and the deregressed breeding values for RE trait, obtained using a ssBLUP model, employed as pseudo-phenotypic data. Our results showed 28 SNPs potentially associated with RE, which explained 87.19% of the genetic variance and 6.61% of the phenotypic variance. Those results were further validated in BayesB, showing a correlation between observed and predicted RE of 0.57. In addition, 15 candidate genes (HTRA3, SPIRE1, APOE, ERCC1, FOXA3, NECTIN-2, KLC3, RSPH6A, PDPK1, MEIOB, PAQR4, NM3, PKD1, PRSS21, IFT140) previously related to fertility in mammals were associated with the markers and genomic regions significantly associated with RE. To our knowledge, this is the first genome-wide association study performed on mare fertility.     

DNA methylation age of human tissues and cell types

Background

It is not yet known whether DNA methylation levels can be used to accurately predict age across a broad spectrum of human tissues and cell types, nor whether the resulting age prediction is a biologically meaningful measure.

Results

I developed a multi-tissue predictor of age that allows one to estimate the DNA methylation age of most tissues and cell types. The predictor, which is freely available, was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. I found that DNA methylation age has the following properties: first, it is close to zero for embryonic and induced pluripotent stem cells; second, it correlates with cell passage number; third, it gives rise to a highly heritable measure of age acceleration; and, fourth, it is applicable to chimpanzee tissues. Analysis of 6,000 cancer samples from 32 datasets showed that all of the considered 20 cancer types exhibit significant age acceleration, with an average of 36 years. Low age-acceleration of cancer tissue is associated with a high number of somatic mutations and TP53 mutations, while mutations in steroid receptors greatly accelerate DNA methylation age in breast cancer. Finally, I characterize the 353 CpG sites that together form an aging clock in terms of chromatin states and tissue variance.

Conclusions

I propose that DNA methylation age measures the cumulative effect of an epigenetic maintenance system. This novel epigenetic clock can be used to address a host of questions in developmental biology, cancer and aging research.     

Optimising experimental design for high-throughput phenotyping in mice: a case study

To further the functional annotation of the mammalian genome, the Sanger Mouse Genetics Programme aims to generate and characterise knockout mice in a high-throughput manner. Annually, approximately 200 lines of knockout mice will be characterised using a standardised battery of phenotyping tests covering key disease indications ranging from obesity to sensory acuity. From these findings secondary centres will select putative mutants of interest for more in-depth, confirmatory experiments. Optimising experimental design and data analysis is essential to maximise output using the resources with greatest efficiency, thereby attaining our biological objective of understanding the role of genes in normal development and disease. This study uses the example of the noninvasive blood pressure test to demonstrate how statistical investigation is important for generating meaningful, reliable results and assessing the design for the defined research objectives. The analysis adjusts for the multiple-testing problem by applying the false discovery rate, which controls the number of false calls within those highlighted as significant. A variance analysis finds that the variation between mice dominates this assay. These variance measures were used to examine the interplay between days, readings, and number of mice on power, the ability to detect change. If an experiment is underpowered, we cannot conclude whether failure to detect a biological difference arises from low power or lack of a distinct phenotype, hence the mice are subjected to testing without gain. Consequently, in confirmatory studies, a power analysis along with the 3Rs can provide justification to increase the number of mice used.     

Characterization of DNA polymeraseβ mutants with amino acid substitutions located in the C-terminal portion of the enzyme

We used quantitative complementation assays to characterize individual DNA polymeraseβ (Polβ) mutants for their ability to function in DNA replication and DNA repair. We also describe a screen for detecting imitator activity of DNA polymeraseβ mutants. By using these bioassays, together with DNA polymerase activity gels, we characterized 15 new DNA polymeraseβ mutants that display a wide spectrum of phenotypes. Most of these mutants are generally defective in their ability to synthesize DNA. However, two of our Polβ mutants show more complex phenotypes: they are able to function in DNA repair but unable to participate in DNA replication. One of our mutants displays imitator activity in vivo. Our work provides a model to study mutant mammalian enzymes inEscherichia coli with phenotypes that are otherwise difficult to assess.     

Estimating mutual information using B-spline functions – an improved similarity measure for analysing gene expression data

Background

The information theoretic concept of mutual information provides a general framework to evaluate dependencies between variables. In the context of the clustering of genes with similar patterns of expression it has been suggested as a general quantity of similarity to extend commonly used linear measures. Since mutual information is defined in terms of discrete variables, its application to continuous data requires the use of binning procedures, which can lead to significant numerical errors for datasets of small or moderate size.

Results

In this work, we propose a method for the numerical estimation of mutual information from continuous data. We investigate the characteristic properties arising from the application of our algorithm and show that our approach outperforms commonly used algorithms: The significance, as a measure of the power of distinction from random correlation, is significantly increased. This concept is subsequently illustrated on two large-scale gene expression datasets and the results are compared to those obtained using other similarity measures.A C++ source code of our algorithm is available for non-commercial use from kloska@scienion.de upon request.

Conclusion

The utilisation of mutual information as similarity measure enables the detection of non-linear correlations in gene expression datasets. Frequently applied linear correlation measures, which are often used on an ad-hoc basis without further justification, are thereby extended.