Exploring possible DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data.

BACKGROUND: Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA sequences. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures. RESULTS: We have developed a new way to examine polymerase kinetics data and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present. CONCLUSION: We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

Promoter microsatellites as modulators of human gene expression.

Microsatellites in and around genes have been shown to modulate levels of gene expression in multiple organisms, ranging from bacteria to humans. Here we will discuss promoter microsatellites known to modulate gene expression, with a few key examples related to the human brain. Many of the microsatellites we discuss are highly conserved in mammals, indicating that selection may favor their retention as "tuning knobs" of gene expression. We will also discuss the mechanisms by which microsatellites in promoters can alter gene expression as they expand and contract, with particular attention to secondary structures like Z-DNA and H-DNA. We suggest that promoter microsatellites, especially those that are highly conserved, may be an important source of human phenotypic variation.

Genetic Information Insecurity as State of the Art.

Genetic information is being generated at an increasingly rapid pace, offering advances in science and medicine that are paralleled only by the threats and risk present within the responsible systems. Human genetic information is identifiable and contains sensitive information, but genetic information security is only recently gaining attention. Genetic data is generated in an evolving and distributed cyber-physical system, with multiple subsystems that handle information and multiple partners that rely and influence the whole ecosystem. This paper characterizes a general genetic information system from the point of biological material collection through long-term data sharing, storage and application in the security context. While all biotechnology stakeholders and ecosystems are valuable assets to the bioeconomy, genetic information systems are particularly vulnerable with great potential for harm and misuse. The security of post-analysis phases of data dissemination and storage have been focused on by others, but the security of wet and dry laboratories is also challenging due to distributed devices and systems that are not designed nor implemented with security in mind. Consequently, industry standards and best operational practices threaten the security of genetic information systems. Extensive development of laboratory security will be required to realize the potential of this emerging field while protecting the bioeconomy and all of its stakeholders.

The genetics of adaptive shape shift in stickleback: pleiotropy and effect size.

The distribution of effect sizes of genes underlying adaptation is unknown (Orr 2005). Are suites of traits that diverged under natural selection controlled by a few pleiotropic genes of large effect (major genes model), by many independently acting genes of small effect (infinitesimal model), or by a combination, with frequency inversely related to effect size (geometric model)? To address this we carried out a quantitative trait loci (QTL) study of a suite of 54 position traits describing body shapes of two threespine stickleback species: an ancestral Pacific marine form and a highly derived benthic species inhabiting a geologically young lake. About half of the 26 detected QTL affected just one coordinate and had small net effects, but several genomic regions affected multiple aspects of shape and had large net effects. The distribution of effect sizes followed the gamma distribution, as predicted by the geometric model of adaptation when detection limits are taken into account. The sex-determining chromosome region had the largest effect of any QTL. Ancestral sexual dimorphism was similar to the direction of divergence, and was largely eliminated during freshwater adaptation, suggesting that sex differences may provide variation upon which selection can act. Several shape QTL are linked to Eda, a major gene responsible for reduction of lateral body armor in freshwater. Our results are consistent with predictions of the geometric model of adaptation. Shape evolution in stickleback results from a few genes with large and possibly widespread effects and multiple genes of smaller effect.

Microsatellite tandem repeats are abundant in human promoters and are associated with regulatory elements.

Tandem repeats are genomic elements that are prone to changes in repeat number and are thus often polymorphic. These sequences are found at a high density at the start of human genes, in the gene's promoter. Increasing empirical evidence suggests that length variation in these tandem repeats can affect gene regulation. One class of tandem repeats, known as microsatellites, rapidly alter in repeat number. Some of the genetic variation induced by microsatellites is known to result in phenotypic variation. Recently, our group developed a novel method for measuring the evolutionary conservation of microsatellites, and with it we discovered that human microsatellites near transcription start sites are often highly conserved. In this study, we examined the properties of microsatellites found in promoters. We found a high density of microsatellites at the start of genes. We showed that microsatellites are statistically associated with promoters using a wavelet analysis, which allowed us to test for associations on multiple scales and to control for other promoter related elements. Because promoter microsatellites tend to be G/C rich, we hypothesized that G/C rich regulatory elements may drive the association between microsatellites and promoters. Our results indicate that CpG islands, G-quadruplexes (G4) and untranslated regulatory regions have highly significant associations with microsatellites, but controlling for these elements in the analysis does not remove the association between microsatellites and promoters. Due to their intrinsic lability and their overlap with predicted functional elements, these results suggest that many promoter microsatellites have the potential to affect human phenotypes by generating mutations in regulatory elements, which may ultimately result in disease. We discuss the potential functions of human promoter microsatellites in this context.

Measuring microsatellite conservation in mammalian evolution with a phylogenetic birth-death model.

Microsatellites make up ∼3% of the human genome, and there is increasing evidence that some microsatellites can have important functions and can be conserved by selection. To investigate this conservation, we performed a genome-wide analysis of human microsatellites and measured their conservation using a binary character birth--death model on a mammalian phylogeny. Using a maximum likelihood method to estimate birth and death rates for different types of microsatellites, we show that the rates at which microsatellites are gained and lost in mammals depend on their sequence composition, length, and position in the genome. Additionally, we use a mixture model to account for unequal death rates among microsatellites across the human genome. We use this model to assign a probability-based conservation score to each microsatellite. We found that microsatellites near the transcription start sites of genes are often highly conserved, and that distance from a microsatellite to the nearest transcription start site is a good predictor of the microsatellite conservation score. An analysis of gene ontology terms for genes that contain microsatellites near their transcription start site reveals that regulatory genes involved in growth and development are highly enriched with conserved microsatellites.