Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized

Nonequilibrium effects in DNA microarrays: a multiplatform study

It has recently been shown that in some DNA microarrays the time needed to reach thermal equilibrium may largely exceed the typical experimental time, which is about 15h in standard protocols (Hooyberghs et al. Phys. Rev. E 81, 012901 (2010)). In this paper we discuss how this breakdown of thermodynamic equilibrium could be detected in microarray experiments without resorting to real time hybridization data, which are difficult to implement in standard experimental conditions. The method is based on the analysis of the distribution of fluorescence intensities I from different spots for probes carrying base mismatches. In thermal equilibrium and at sufficiently low concentrations, log I is expected to be linearly related to the hybridization free energy $\Delta G$ with a slope equal to $1/RT_{exp}$, where $T_{exp}$ is the experimental temperature and R is the gas constant. The breakdown of equilibrium results in the deviation from this law. A model for hybridization kinetics explaining the observed experimental behavior is discussed, the so-called 3-state model. It predicts that deviations from equilibrium yield a proportionality of $\log I$ to $\Delta G/RT_{eff}$. Here, $T_{eff}$ is an effective temperature, higher than the experimental one. This behavior is indeed observed in some experiments on Agilent arrays. We analyze experimental data from two other microarray platforms and discuss, on the basis of the results, the attainment of equilibrium in these cases. Interestingly, the same 3-state model predicts a (dynamical) saturation of the signal at values below the expected one at equilibrium.Comment: 27 pages, 9 figures, 1 tabl

Tapping culture collections for fungal endophytes: first genome assemblies for three genera and five species in the Ascomycota

The Ascomycota form the largest phylum in the fungal kingdom and show a wide diversity of lifestyles, some involving associations with plants. Genomic data are available for many ascomycetes that are pathogenic to plants, but endophytes, which are asymptomatic inhabitants of plants, are relatively understudied. Here, using short- and long-read technologies, we have sequenced and assembled genomes for 15 endophytic ascomycete strains from CABI’s culture collections. We used phylogenetic analysis to refine the classification of taxa, which revealed that 7 of our 15 genome assemblies are the first for the genus and/or species. We also demonstrated that cytometric genome size estimates can act as a valuable metric for assessing assembly “completeness”, which can easily be overestimated when using BUSCOs alone and has broader implications for genome assembly initiatives. In producing these new genome resources, we emphasise the value of mining existing culture collections to produce data that can help to address major research questions relating to plant–fungal interactions

Arrested spinodal decomposition in polymer brush collapsing in poor solvent

We study the Brownian dynamics of flexible and semiflexible polymer chains densely grafted on a flat substrate, upon rapid quenching of the system when the quality of solvent becomes poor and chains attempt collapse into a globular state. The collapse process of such a polymer brush differs from individual chains, both in its kinetics and its structural morphology. We find that the resulting collapsed brush does not form a homogeneous dense layer, in spite of all chain monomers equally attracting each other via a model Lennard-Jones potential. Instead, a very distinct inhomogeneous density distribution in the plane forms, with a characteristic length scale dependent on the quenching depth (or equivalently, the strength of monomer attraction) and the geometric parameters of the brush. This structure is identical to the spinodal-decomposition structure, however, due to the grafting constraint we find no subsequent coarsening: the established random bundling with characteristic periodicity remains as the apparently equilibrium structure. We compare this finding with a recent field-theoretical model of bundling in a semiflexible polymer brush.This work was funded by the Osk. Huttunen Foundation (Finland) and the Cambridge Theory of Condensed Matter Grant from EPSRC. Simulations were performed using the Darwin supercomputer of the University of Cambridge High Performance Computing Service provided by Dell Inc. using Strategic Research Infrastructure funding from the Higher Education Funding Council for England.This is the accepted manuscript. The final version is available at http://pubs.acs.org/doi/abs/10.1021/ma501985r

SERS-melting: a new method for discriminating mutations in dna sequences

The reliable discrimination of mutations, single nucleotide polymorphisms (SNPs), and other differences in genomic sequence is an essential part of DNA diagnostics and forensics. It is commonly achieved using fluorescently labeled DNA probes and thermal gradients to distinguish between the matched and mismatched DNA. Here, we describe a novel method that uses surface enhanced (resonance) Raman spectroscopy (SER(R)S) to follow denaturation of dsDNA attached to a structured gold surface. This denaturation is driven either electrochemically or thermally on SERS active sphere segment void (SSV) gold substrates. Using this method, we can distinguish between wild type, a single point mutation (1653C/T), and a triple deletion (?F 508) in the CFTR gene at the 0.02 attomole level, and the method can be used to differentiate the unpurified PCR products of the wild type and ?F 508 mutation. Our method has the potential to provide small, rapid, sensitive, reproducible platforms for detecting genetic variations and sequencing genes

Robust Digital Watermarking Based on the Log-Polar Mapping

The geometrical attacks are still an open problem for many digital watermarking algorithms used in present time. Most of geometrical attacks can be described by using affine transforms. This article deals with digital watermarking in images robust against the affine transformations. The new approach to improve robustness against geometrical attacks is presented. The discrete Fourier transform and log-polar mapping is used for watermark embedding and for watermark detection. Some attacks against the embedded watermarks are performed and the results are given

Some Modifications of Fractal Image Coding

In this paper some modifications of fractal image coding are presented. Proposed methods are based on correlation coefficients computing as an alternative approach to searching of similarity between blocks. The convergence speed of decoding process is faster then convergence speed of standard method. The convergence process with modified start conditions of decoding process are analysed and verified on gray scale static images too