Software for optimization of SNP and PCR-RFLP genotyping to discriminate many genomes with the fewest assays

BACKGROUND: Microbial forensics is important in tracking the source of a pathogen, whether the disease is a naturally occurring outbreak or part of a criminal investigation. RESULTS: A method and SPR Opt (SNP and PCR-RFLP Optimization) software to perform a comprehensive, whole-genome analysis to forensically discriminate multiple sequences is presented. Tools for the optimization of forensic typing using Single Nucleotide Polymorphism (SNP) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses across multiple isolate sequences of a species are described. The PCR-RFLP analysis includes prediction and selection of optimal primers and restriction enzymes to enable maximum isolate discrimination based on sequence information. SPR Opt calculates all SNP or PCR-RFLP variations present in the sequences, groups them into haplotypes according to their co-segregation across those sequences, and performs combinatoric analyses to determine which sets of haplotypes provide maximal discrimination among all the input sequences. Those set combinations requiring that membership in the fewest haplotypes be queried (i.e. the fewest assays be performed) are found. These analyses highlight variable regions based on existing sequence data. These markers may be heterogeneous among unsequenced isolates as well, and thus may be useful for characterizing the relationships among unsequenced as well as sequenced isolates. The predictions are multi-locus. Analyses of mumps and SARS viruses are summarized. Phylogenetic trees created based on SNPs, PCR-RFLPs, and full genomes are compared for SARS virus, illustrating that purported phylogenies based only on SNP or PCR-RFLP variations do not match those based on multiple sequence alignment of the full genomes. CONCLUSION: This is the first software to optimize the selection of forensic markers to maximize information gained from the fewest assays, accepting whole or partial genome sequence data as input. As more sequence data becomes available for multiple strains and isolates of a species, automated, computational approaches such as those described here will be essential to make sense of large amounts of information, and to guide and optimize efforts in the laboratory. The software and source code for SPR Opt is publicly available and free for non-profit use at

CRASH: A real-time 3D game engine designed to make game development easy

Developing games on top of commercial game engines is difficult because the projects are too large to quickly understand. We present Crash: an alternative solution to game development that empowers developers to begin working on a game with very little introduction to the project by building a small, extensible, and modular game engine. Design patterns such as dependency injection and interface-based development encourage simple, understandable code and empower developers to divert their efforts toward the playable components of their games

A Novel Method for Quantifying Spatial Patterns in Plants

Color patterns are found in a plethora of organisms, from vertebrates to flowering plants. While many studies have examined the mechanisms that produce these diverse patterns in animals, little research has investigated the mechanisms by which plants create color patterns. The conclusions drawn from animal studies may not accurately translate to plants due to early divergence in the evolution of life. Characterization of plant patterning mechanisms would have widespread impacts on developmental and evolutionary biology. To unravel the mystery behind pattern formation, we suggest an experimental framework to understand pattern evolution and development at a phenotypic, genotypic, and quantitative level, creating a holistic model for the evolution of complex traits and phenotypic diversity. Here, we provide a novel protocol for the quantification of pattern morphology, and demonstrate its efficacy in a segregating F2 population of the model organism Mimulus luteus . By co-opting ArcGIS and FragStats, two landscape ecology softwares, to map petal patterns, we developed a high throughput method for objective phenotype characterization. This protocol is useful for preliminary work in a bulk segregant analysis by separating a population in discrete groups based on morphology. We used this protocol to demonstrate that patterns are distinct between petals within the same flower depending on petal location, and that there is a genetic basis for pattern formation in flowers. Minor tweaks to the genes guiding pattern formation may be responsible for the rapid evolution of angiosperm flower diversity. Future work is required to identify the genes responsible for pattern formation, and to develop a method for modeling these genes to predict how minor mutations would impact phenotypic traits

Mutations causing medullary cystic kidney disease type 1 lie in a large VNTR in MUC1 missed by massively parallel sequencing

Although genetic lesions responsible for some mendelian disorders can be rapidly discovered through massively parallel sequencing of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing and de novo assembly did we find that each of six families with MCKD1 harbors an equivalent but apparently independently arising mutation in sequence markedly under-represented in massively parallel sequencing data: the insertion of a single cytosine in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5–5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1. These results provide a cautionary tale about the challenges in identifying the genes responsible for mendelian, let alone more complex, disorders through massively parallel sequencing.National Institutes of Health (U.S.) (Intramural Research Program)National Human Genome Research Institute (U.S.)Charles University (program UNCE 204011)Charles University (program PRVOUK-P24/LF1/3)Czech Republic. Ministry of Education, Youth, and Sports (grant NT13116-4/2012)Czech Republic. Ministry of Health (grant NT13116-4/2012)Czech Republic. Ministry of Health (grant LH12015)National Institutes of Health (U.S.) (Harvard Digestive Diseases Center, grant DK34854

A neuronal device for the control of multi-step computations

We describe the operation of a neuronal device which embodies the computational principles of the `paper-and-pencil' machine envisioned by Alan Turing. The network is based on principles of cortical organization. We develop a plausible solution to implement pointers and investigate how neuronal circuits may instantiate the basic operations involved in assigning a value to a variable (i.e., x=5), in determining whether two variables have the same value and in retrieving the value of a given variable to be accessible to other nodes of the network. We exemplify the collective function of the network in simplified arithmetic and problem solving (blocks-world) tasks.Fil: Zylberberg, Ariel Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación. Laboratorio de Inteligencia Artificial y Aplicaciones; ArgentinaFil: Paz, Luciano. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Neurociencia Integrativa; ArgentinaFil: Roelfsema, Pieter R.. Commissariat A Energie Atomique; FranciaFil: Dehaene, Stanislas. Commissariat A Energie Atomique; FranciaFil: Sigman, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; Argentin

How to perform RT-qPCR accurately in plant species?: a case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

Background: Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. Results: An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. Conclusions: Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway

Continuous powder mixing of segregating mixtures under steady and unsteady state regimes: Homogeneity assessment by real-time on-line image analysis

Continuous powder processes, such as continuous powder mixing, are more than ever envisioned as a viable alternative to batch equipment, in various industries such as pharmaceuticals, specialty chemicals (zeolites, SiC), bio-renewables or food. In the present work we have implemented an on-line image analysis set-up that is able to capture all the images of the particles at the outlet of a continuous pilot-scale mixer. This allows the determination of the homogeneity of mixtures of two different compositions, as well as the analysis of their evolution during steady-state and transitory regimes. The importance of a proper definition of the scale of scrutiny of the mixture is emphasized by providing homogeneity results obtained at four different scales. Evidence of segregation by percolation giving rise to the enrichment of the mixer's bed with fine particles is given and commented. The impact of the stirrer's rotational speed on the quality of the mixtures, as provided by the coefficient of variation CV, is reported. Up to 20 Hz, CV's are extremely high, while above 30 Hz, the influence of the impeller speed is much weaker. Finally, the influence of impeller speed's step perturbations is measured and commented. Due to the size – segregation phenomenon inside the mixer, negative steps are deeply detrimental to the mixing process. On the contrary, positive steps can be absorbed by the equipment without degradation of the quality of the mixtures

Divergent T-fO2 paths during crystallisation of H2O-rich and H2O-poor magmas as recorded by Ce and U in zircon, with implications for TitaniQ and TitaniZ geothermometry

During solidification of magma chambers as systems closed to chemical exchange with environs, the residual siliceous melt may follow a trend of rising, constant, or decreasing oxidation state, relative to reference buffers such as nickel + nickel oxide (NNO) or fayalite + magnetite + quartz. Titanomagnetite–hemoilmenite thermometry and oxybarometry on quenched volcanic suites yield temperature versus oxygen fugacity arrays of varied positive and negative slopes, the validity of which has been disputed for several years. We resolve the controversy by introducing a new recorder of magmatic redox evolution employing temperature- and redox-sensitive trace-element abundances in zircon. The zircon/melt partition coefficients of cerium and uranium vary oppositely in response to variation of magma redox state, but vary in tandem as temperature varies. Plots of U/Pr versus Ce4+/Ce3+ in zircon provide a robust test for change in oxidation state of the melt during zircon crystallisation from cooling magma, and the plots discriminate thermally induced from redox-induced variation of Ce4+/Ce3+ in zircon. Temperature-dependent lattice strain causes Ce4+/Ce3+ in zircon to increase strongly as zircon crystallises from cooling magma at constant Ce4+/Ce3+ ratio in the melt. We examine 19 zircon populations from igneous complexes in varied tectonic settings. Variation of zircon Ce4+/Ce3+ due to minor variation in melt oxidation state during crystallisation is resolvable in 11 cases but very subordinate to temperature dependence. In many zircon populations described in published literature, there is no resolvable change in redox state of the melt during tenfold variation of Ce4+/Ce3+ in zircons. Varied magmatic redox trends indicated by different slopes on plots of zircon U/Pr versus Ce4+/Ce3+ are corroborated by Fe–Ti-oxide-based T–ƒO2 trends of correspondingly varied slopes. Zircon and Fe–Ti-oxide compositions agree that exceptionally, H2O-rich arc magmas tend to follow a trend of rising oxidation state of the melt during late stages of fluid-saturated magmatic differentiation at upper-crustal pressures. We suggest that H2 and/or SO3 and/or Fe2+ loss from the melt to segregating fluid is largely responsible. Conversely, zircon and Fe–Ti-oxide compositions agree in indicating that H2O-poor magmas tend to follow a T–ƒO2 trend of decreasing oxidation state of the melt during late stages of magmatic differentiation at upper-crustal pressures, because the precipitating mineral assemblage has higher Fe3+/Fe2+ than coexisting rhyolitic melt. We present new evidence showing that the Fe–Ti-oxide oxybarometer calibration by Ghiorso and Evans (Am J Sci 308(9):957–1039, 2008) retrieves experimentally imposed values of ƒO2 in laboratory syntheses of Fe–Ti-oxide pairs to a precision of ± 0.2 log unit, over a large experimental temperature range, without systematic bias up to at least log ƒO2 ≈ NNO + 4.4. Their titanomagnetite–hemoilmenite geothermometer calibration has large systematic errors in application to Ti-poor oxides that precipitate from very oxidised magmas. A key outcome is validation of Fe–Ti-oxide-based values of melt TiO2 activity for use in Ti-in-zircon thermometry and Ti-in-quartz thermobarometry.We thank Paul Agnew and Alan Kobussen of Rio Tinto Exploration for financial support and for authorisation to publish research results. Additional financial support was provided by the ARC Centre of Excellence for Core to Crust Fluid Systems Grant CE110001017. MLF acknowledges the support of the Australian Research Council through the Future Fellowship Grant scheme (FT110100241). MLF also acknowledges support from the Australian Research Council through Linkage Project LP120100668. BDR acknowledges WMC Resources Ltd for financial support of his PhD research. RRL thanks the University of Bristol for a Benjamin Meaker Visiting Professorship which afforded an opportunity to clarify his understanding of this material through discussions with Professors Jon Blundy and Chris Hawkesworth