Sequence verification of synthetic DNA by assembly of sequencing reads

Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Sequence validation is equally important for other kinds of curated clone collections. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. GenoREAD can determine if a clone matches its reference sequence. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD is available at www.genoread.or

Deterministic and stochastic descriptions of gene expression dynamics

A key goal of systems biology is the predictive mathematical description of gene regulatory circuits. Different approaches are used such as deterministic and stochastic models, models that describe cell growth and division explicitly or implicitly etc. Here we consider simple systems of unregulated (constitutive) gene expression and compare different mathematical descriptions systematically to obtain insight into the errors that are introduced by various common approximations such as describing cell growth and division by an effective protein degradation term. In particular, we show that the population average of protein content of a cell exhibits a subtle dependence on the dynamics of growth and division, the specific model for volume growth and the age structure of the population. Nevertheless, the error made by models with implicit cell growth and division is quite small. Furthermore, we compare various models that are partially stochastic to investigate the impact of different sources of (intrinsic) noise. This comparison indicates that different sources of noise (protein synthesis, partitioning in cell division) contribute comparable amounts of noise if protein synthesis is not or only weakly bursty. If protein synthesis is very bursty, the burstiness is the dominant noise source, independent of other details of the model. Finally, we discuss two sources of extrinsic noise: cell-to-cell variations in protein content due to cells being at different stages in the division cycles, which we show to be small (for the protein concentration and, surprisingly, also for the protein copy number per cell) and fluctuations in the growth rate, which can have a significant impact.Comment: 23 pages, 5 figures; Journal of Statistical physics (2012

Investigar en tiempos de aislamiento social. Decisiones metodológicas en una investigación en curso

En el artículo se reflexiona sobre el alcance epistemológico de la incorporación de soportes tecnológicos para la recolección de datos y realización de entrevistas en profundidad en una investigación de tesis doctoral en curso afectada en su continuidad por la pandemia de COVID-19. Se pone a disposición el análisis sobre las posibilidades y límites de los medios digitales y nuevas tecnologías para garantizar la integridad de los datos al momento de incorporar modificaciones en el proceso de recolección de información y se explicitan las decisiones asumidas en torno a las herramientas y modalidad de recolección de datos para continuar con la investigación doctoral. Palabras clave: investigación cualitativa, investigación electrónica, investigación en pandemia, entornos virtuales, técnicas de recolección de datos

Effect of promoter architecture on the cell-to-cell variability in gene expression

According to recent experimental evidence, the architecture of a promoter, defined as the number, strength and regulatory role of the operators that control the promoter, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect noise in gene expression in a systematic rather than case-by-case fashion. In this article, we make such a systematic investigation, based on a simple microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous) and how each of these affects the level of variability in transcription product from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can discriminate between different kinetic models of gene regulation.Comment: 35 pages, 6 figures, Submitte

Sequence verification of synthetic DNA by assembly of sequencing reads

This is the publisher’s final pdf. The published article is copyrighted by Oxford University Press and can be found at: http://www.oxfordjournals.org/Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Sequence validation is equally important for other kinds of curated clone collections. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. GenoREAD can determine if a clone matches its reference sequence. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD is available at www.genoread.org

GenoCAD for iGEM: a grammatical approach to the design of standard-compliant constructs

One of the foundations of synthetic biology is the project to develop libraries of standardized genetic parts that could be assembled quickly and cheaply into large systems. The limitations of the initial BioBrick standard have prompted the development of multiple new standards proposing different avenues to overcome these shortcomings. The lack of compatibility between standards, the compliance of parts with only some of the standards or even the type of constructs that each standard supports have significantly increased the complexity of assembling constructs from standardized parts. Here, we describe computer tools to facilitate the rigorous description of part compositions in the context of a rapidly changing landscape of physical construction methods and standards. A context-free grammar has been developed to model the structure of constructs compliant with six popular assembly standards. Its implementation in GenoCAD makes it possible for users to quickly assemble from a rich library of genetic parts, constructs compliant with any of six existing standards

The chemical signatures underlying host plant discrimination by aphids

The diversity of phytophagous insects is largely attributable to speciation involving shifts between host plants. These shifts are mediated by the close interaction between insects and plant metabolites. However, there has been limited progress in understanding the chemical signatures that underlie host preferences. We use the pea aphid (Acyrthosiphon pisum) to address this problem. Host-associated races of pea aphid discriminate between plant species in race-specific ways. We combined metabolomic profiling of multiple plant species with behavioural tests on two A. pisum races, to identify metabolites that explain variation in either acceptance or discrimination. Candidate compounds were identified using tandem mass spectrometry. Our results reveal a small number of compounds that explain a large proportion of variation in the differential acceptability of plants to A. pisum races. Two of these were identified as L-phenylalanine and L-tyrosine but it may be that metabolically-related compounds directly influence insect behaviour. The compounds implicated in differential acceptability were not related to the set correlated with general acceptability of plants to aphids, regardless of host race. Small changes in response to common metabolites may underlie host shifts. This study opens new opportunities for understanding the mechanistic basis of host discrimination and host shifts in insects

Tipping points in the dynamics of speciation.

Speciation can be gradual or sudden and involve few or many genetic changes. Inferring the processes generating such patterns is difficult, and may require consideration of emergent and non-linear properties of speciation, such as when small changes at tipping points have large effects on differentiation. Tipping points involve positive feedback and indirect selection stemming from associations between genomic regions, bi-stability due to effects of initial conditions and evolutionary history, and dependence on modularity of system components. These features are associated with sudden 'regime shifts' in other cellular, ecological, and societal systems. Thus, tools used to understand other complex systems could be fruitfully applied in speciation research

Sequence verification of synthetic DNA by assembly of sequencing reads

Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Sequence validation is equally important for other kinds of curated clone collections. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. GenoREAD can determine if a clone matches its reference sequence. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD is available at www.genoread.org.This is the publisher’s final pdf. The published article is copyrighted by Oxford University Press and can be found at: http://www.oxfordjournals.org/Keywords: Bioinformatics, Gene synthesis, Genome, Retrieval, Biology, Alignmen

Balancing Robustness against the Dangers of Multiple Attractors in a Hopfield-Type Model of Biological Attractors

Background: Many chronic human diseases are of unclear origin, and persist long beyond any known insult or instigating factor. These diseases may represent a structurally normal biologic network that has become trapped within the basin of an abnormal attractor. Methodology/Principal Findings: We used the Hopfield net as the archetypical example of a dynamic biological network. By progressively removing the links of fully connected Hopfield nets, we found that a designated attractor of the nets could still be supported until only slightly more than 1 link per node remained. As the number of links approached this minimum value, the rate of convergence to this attractor from an arbitrary starting state increased dramatically. Furthermore, with more than about twice the minimum of links, the net became increasingly able to support a second attractor. Conclusions/Significance: We speculate that homeostatic biological networks may have evolved to assume a degree of connectivity that balances robustness and agility against the dangers of becoming trapped in an abnormal attractor