Large scale loss of data in low-diversity illumina sequencing libraries can be recovered by deferred cluster calling

Massively parallel DNA sequencing is capable of sequencing tens of millions of DNA fragments at the same time. However, sequence bias in the initial cycles, which are used to determine the coordinates of individual clusters, causes a loss of fidelity in cluster identification on Illumina Genome Analysers. This can result in a significant reduction in the numbers of clusters that can be analysed. Such low sample diversity is an intrinsic problem of sequencing libraries that are generated by restriction enzyme digestion, such as e4C-seq or reduced-representation libraries. Similarly, this problem can also arise through the combined sequencing of barcoded, multiplexed libraries. We describe a procedure to defer the mapping of cluster coordinates until low-diversity sequences have been passed. This simple procedure can recover substantial amounts of next generation sequencing data that would otherwise be lost.     

Technical Factors Influencing Cone Packing Density Estimates in Adaptive Optics Flood Illuminated Retinal Images

Purpose

To investigate the influence of various technical factors on the variation of cone packing density estimates in adaptive optics flood illuminated retinal images.

Methods

Adaptive optics images of the photoreceptor mosaic were obtained in fifteen healthy subjects. The cone density and Voronoi diagrams were assessed in sampling windows of 320×320 µm, 160×160 µm and 64×64 µm at 1.5 degree temporal and superior eccentricity from the preferred locus of fixation (PRL). The technical factors that have been analyzed included the sampling window size, the corrected retinal magnification factor (RMF_corr), the conversion from radial to linear distance from the PRL, the displacement between the PRL and foveal center and the manual checking of cone identification algorithm. Bland-Altman analysis was used to assess the agreement between cone density estimated within the different sampling window conditions.

Results

The cone density declined with decreasing sampling area and data between areas of different size showed low agreement. A high agreement was found between sampling areas of the same size when comparing density calculated with or without using individual RMF_corr. The agreement between cone density measured at radial and linear distances from the PRL and between data referred to the PRL or the foveal center was moderate. The percentage of Voronoi tiles with hexagonal packing arrangement was comparable between sampling areas of different size. The boundary effect, presence of any retinal vessels, and the manual selection of cones missed by the automated identification algorithm were identified as the factors influencing variation of cone packing arrangements in Voronoi diagrams.

Conclusions

The sampling window size is the main technical factor that influences variation of cone density. Clear identification of each cone in the image and the use of a large buffer zone are necessary to minimize factors influencing variation of Voronoi diagrams of the cone mosaic.     

Estimating subject-specific body segment parameters using a 3-dimensional modeller program

The estimation of body segment properties is important in the biomechanical analysis of movement. Current subject-specific estimation methods however can be expensive and time-consuming, while other methods do not adequately take into account individual or group variability. We describe a simple procedure for estimating subject-specific geometric properties, independent of joint centres. The method requires only a small number of anthropometric measurements and digital images of the segment or subject, a 3-dimensional modeller program and simple mathematical calculations to estimate segment volumes and centroids. Assuming that the segment is of uniform density, it's mass and moment of inertia can also be derived. Future work should include generating segment density profiles for particular populations, to increase the accuracy of the method, and comparing the accuracy of the results obtained with those produced by other techniques.     

Voro3D: 3D Voronoi tessellations applied to protein structures

SUMMARY: Voro3D is an original easy-to-use tool, which provides a brand new point of view on protein structures through the three-dimensional (3D) Voronoi tessellations. To construct the Voronoi cells associated with each amino acid by a number of different tessellation methods, Voro3D uses a protein structure file in the PDB format as an input. After calculation, different structural properties of interest like secondary structures assignment, environment accessibility and exact contact matrices can be derived without any geometrical cut-off. Voro3D provides also a visualization of these tessellations superimposed on the associated protein structure, from which it is possible to model a polygonal protein surface using a model solvent or to quantify, for instance, the contact areas between a protein and a ligand. AVAILABILITY: The software executable file for PC using Windows 98, 2000, NT, XP can be freely downloaded at http://www.lmcp.jussieu.fr/~mornon/voronoi.html CONTACT: franck.dupuis@sanofi-aventis.com; jean-paul-mornon@imcp.jussieu.fr.     

Quantification of protein surfaces,volumes and atom-atom contacts using a constrained Voronoi procedure

MOTIVATION: Geometric representations of proteins and ligands, including atom volumes, atom-atom contacts and solvent accessible surfaces, can be used to characterize interactions between and within proteins, ligands and solvent. Voronoi algorithms permit quantification of these properties by dividing structures into cells with a one-to-one correspondence with constituent atoms. As there is no generally accepted measure of atom-atom contacts, a continuous analytical representation of inter-atomic contacts will be useful. Improved geometric algorithms will also be helpful in increasing the speed and accuracy of iterative modeling algorithms. RESULTS: We present computational methods based on the Voronoi procedure that provide rapid and exact solutions to solvent accessible surfaces, volumes, and atom contacts within macromolecules. Furthermore, we define a measure of atom-atom contact that is consistent with the calculation of solvent accessible surfaces, allowing the integration of solvent accessibility and inter-atomic contacts into a continuous measure. The speed and accuracy of the algorithm is compared to existing methods for calculating solvent accessible surfaces and volumes. The presented algorithm has a reduced execution time and greater accuracy compared to numerical and approximate analytical surface calculation algorithms, and a reduced execution time and similar accuracy to existing Voronoi procedures for calculating atomic surfaces and volumes.     

Determination of cellulose crystallinity from powder diffraction diagrams

One‐dimensional (1D) (spherically averaged) powder diffraction diagrams are commonly used to determine the degree of cellulose crystallinity in biomass samples. Here, it is shown using molecular modeling how disorder in cellulose fibrils can lead to considerable uncertainty in conclusions drawn concerning crystallinity based on 1D powder diffraction data alone. For example, cellulose microfibrils that contain both crystalline and noncrystalline segments can lead to powder diffraction diagrams lacking identifiable peaks, while microfibrils without any crystalline segments can lead to such peaks. This leads to false positives, that is, assigning disordered cellulose as crystalline, and false negatives, that is, categorizing fibrils with crystalline segments as amorphous. The reliable determination of the fraction of crystallinity in any given biomass sample will require a more sophisticated approach combining detailed experiment and simulation. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 67–73, 2015.     

The Obstacle-Set Method for Representing Muscle Paths in Musculoskeletal Models

A computational method is introduced for modeling the paths of muscles in the human body. The method is based on the premise that the resultant muscle force acts along the locus of the transverse cross-sectional centroids of the muscle. The path of the muscle is calculated by idealizing its centroid path as a frictionless elastic band, which moves freely over neighboring anatomical constraints such as bones and other muscles. The anatomical constraints, referred to as obstacles, are represented in the model by regular-shaped, rigid bodies such as spheres and cylinders. The obstacles, together with the muscle path, define an obstacle set. It is proposed that the path of any muscle can be modeled using one or more of the following four obstacle sets: single sphere, single cylinder, double cylinder, and sphere-capped cylinder. Assuming that the locus of the muscle centroids is known for an arbitrary joint configuration, the obstacle-set method can be used to calculate the path of the muscle for all other joint configurations. The obstacle-set method accounts not only for the interaction between a muscle and a neighboring anatomical constraint, but also for the way in which this interaction changes with joint configuration. Consequently, it is the only feasible method for representing the paths of muscles which cross joints with multiple degrees of freedom such as the deltoid at the shoulder.     

Dynamic metabolic modeling for a MAB bioprocess

Production of monoclonal antibodies (MAb) for diagnostic or therapeutic applications has become an important task in the pharmaceutical industry. The efficiency of high-density reactor systems can be potentially increased by model-based design and control strategies. Therefore, a reliable kinetic model for cell metabolism is required. A systematic procedure based on metabolic modeling is used to model nutrient uptake and key product formation in a MAb bioprocess during both the growth and post-growth phases. The approach combines the key advantages of stoichiometric and kinetic models into a complete metabolic network while integrating the regulation and control of cellular activity. This modeling procedure can be easily applied to any cell line during both the cell growth and post-growth phases. Quadratic programming (QP) has been identified as a suitable method to solve the underdetermined constrained problem related to model parameter identification. The approach is illustrated for the case of murine hybridoma cells cultivated in stirred spinners.     

Proportion estimation with confidence limits

A common task in microbiology involves determining the composition of a mixed population of individuals by drawing a sample from the population and using some procedure to identify the individuals in the sample. There may be a significant probability that the identification procedure misidentifies some members of the sample (for example, because the available data are insufficient unambiguously to identify an individual) which makes finding the proportions in the underlying population non-trivial. A further complication arises where individuals are present in the population that do not belong to any of the subpopulations recognised by use of the identification procedure. A simple algorithm is presented to address these problems and construct a maximum likelihood estimate of the proportions, together with confidence limits. The technique is illustrated using an example drawn from flow cytometry in which phytoplankton cells are identified from flow cytometry data by an RBF neural network, and the limitations of the approach are discussed.     

Automatic image segmentation with linear clustering for quantification of neointimal formation after surgical vein grafting

OBJECTIVE: To identify extracellular matrix deposition on combined Masson elastin stains from cross-sectional, fixed vein grafts. STUDY DESIGN: Source vectors from RGB components of color images are transformed into new vectors with most of the energy concentrated in fewer coefficients based on the eigenvalues and eigenvectors of their co-variance matrix so their dimension can be reduced for efficient computation and analysis. The vectors are distributed in a triangular shape in which most vectors are located in a long, narrow strip that can be approximated by a straight line while a separate group of vectors from collagen areas form a loose cluster away from the line. An iterative procedure has been developed for the representative vectors in the 2 centroids for linear and circular clusters. The linear centroid consists of all vectors in a straight line, and the centroid of the circular cluster is a single vector. Vector classification is based on the measure of its distance to each of the 2 centroids. RESULTS: The automatic segmentation of the collagen content pixels in green-blue matches the image background color. CONCLUSION: The procedure automatically quantifies and characterizes the neointimal deposition after surgical vein grafting in mice.     

HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures

Criteria for scoring micronuclei and nucleoplasmic bridges in binucleated cells in the cytokinesis-block micronucleus assay for isolated human lymphocyte cultures are described in detail. Morphological characteristics of mononucleated cells, binucleated cells, and multinucleated cells as well as necrotic and apoptotic cells and nuclear buds are also described. These criteria are illustrated by a series of schematic diagrams as well as a comprehensive set of colour photographs that are of practical assistance during the scoring of slides. These scoring criteria, diagrams and photographs have been used in a HUman MicronNucleus (HUMN) project inter-laboratory slide-scoring exercise to evaluate the extent of variability that can be attributable to individual scorers and individual laboratories when measuring the frequency of micronuclei and nucleoplasmic bridges in binucleated cells as well as the nuclear division index. The results of the latter study are described in an accompanying paper. It is expected that these scoring criteria will assist in the development of a procedure for calibrating scorers and laboratories so that results from different laboratories for the cytokinesis-block micronucleus assay may be more comparable in the future.     

A method for analysing spatial scales of variation in composition of assemblages

In several areas of research on ecological assemblages, it is useful to be able to analyse patterns of spatial variation at various scales. Multivariate analyses of dissimilarity or similarity in assemblages of species are limited by problems of non-independence caused by repeated use of the sample-units. Where rank-order procedures are used, no comparative quantitative measurements of dissimilarity at different scales are produced. An alternative method is described that uses the sample's average assemblage (or centroid). These estimates are themselves averaged to give centroids for larger spatial scales. Dissimilarities from the centroids at each scale are then calculated using independent replicates for each scale from those in each sample. The dissimilarity measures can then be examined by analysis of variance to detect spatial scales of differences for each sample at every level of a hierarchy of scales. The method is illustrated using data from mangrove forests and rocky shores, involving up to 97 taxonomic groups (species, other taxa). Differences among assemblages at the scales of sites (tens of meters apart) or locations at shores (hundreds of meters apart) were identified. Consequences of different numbers of replicates are discussed, with some potential problems (and their solutions) in application. Received: 14 November 1997 / Accepted: 14 September 1998     

Using Publicly Available Forest Inventory Data in Climate-Based Models of Tree Species Distribution: Examining Effects of True Versus Altered Location Coordinates

Species distribution models (SDMs) were built with US Forest Inventory and Analysis (FIA) publicly available plot coordinates, which are altered for plot security purposes, and compared with SDMs built with true plot coordinates. Six species endemic to the western US, including four junipers (Juniperus deppeana var. deppeana, J. monosperma, J. occidentalis, J. osteosperma) and two piñons (Pinus edulis, P. monophylla), were analyzed. The presence–absence models based on current climatic variables were generated over a series of species-specific modeling extents using Random Forests and applied to forecast climatic conditions. The distributions of predictor variables sampled with public coordinates were compared to those sampled with true coordinates using t tests with a Bonferroni adjustment for multiple comparisons. Public- and true-based models were compared using metrics of classification accuracy. The modeled current and forecast distributions were compared in terms of their overall areal agreement and their geographic mean centroids. Comparison of the underlying distributions of predictor variables sampled with true versus public coordinates did not indicate a significant difference for any species at any extent. Both the public- and true-based models had comparable classification accuracies across extent for each species, with the exception of one species, J. occidentalis. True-based models produced geographic distributions with smaller areas under current and future scenarios. The greatest areal difference occurred in the species with the lowest modeled accuracies (J. occidentalis), and had a forecast distribution which diverged severely. The other species had forecast distributions with similar magnitudes of modeled distribution shifts.     

Building proteins from C alpha coordinates using the dihedral probability grid Monte Carlo method.

Dihedral probability grid Monte Carlo (DPG-MC) is a general-purpose method of conformational sampling that can be applied to many problems in peptide and protein modeling. Here we present the DPG-MC method and apply it to predicting complete protein structures from C alpha coordinates. This is useful in such endeavors as homology modeling, protein structure prediction from lattice simulations, or fitting protein structures to X-ray crystallographic data. It also serves as an example of how DPG-MC can be applied to systems with geometric constraints. The conformational propensities for individual residues are used to guide conformational searches as the protein is built from the amino-terminus to the carboxyl-terminus. Results for a number of proteins show that both the backbone and side chain can be accurately modeled using DPG-MC. Backbone atoms are generally predicted with RMS errors of about 0.5 A (compared to X-ray crystal structure coordinates) and all atoms are predicted to an RMS error of 1.7 A or better.     

Conformational studies of nucleic acids. I. A rapid and direct method for generating furanose coordinates from the pseudorotation angle

The greatest difficulty in modeling a nucleic acid is generating the coordinates of its furanoses. This difficulty arises from constraints imposed by the closed ring geometries of these sugars. We have developed a new method for modeling these furanose rings. Using this method, the coordinates of a sugar can be obtained quickly and unambiguously for any point on the pseudorotational pathway from one parameter: the phase angle of pseudorotation P. The significant difference between this and previous sugar modeling schemes is that here the endocyclic bond lengths of the five-membered sugar ring are allowed to vary a small amount according to simple, explicit, and experimentally reasonable analytic functions of P. The coefficients of these functions follow from the empirical behavior of the endocyclic bond angles and from geometrical constraints due to ring closure. The ability to model the sugars directly from one parameter greatly facilitates carrying out the global conformational studies on nucleic acid constituents which will be attempted in subsequent papers of this series.     

Freeze substitution of fungi for cytological analysis

The preparatory techniques of freeze substitution for electron microscopy are given in detail. Included is a simple, reliable method for the selection of individual freeze-substituted cells from flat embedments via light microscopy for subsequent electron microscope analysis. These mthods are intended for studying cells in monolayer cultures, tissues, suspensions of cells, or cell fractions and are widely applicable for cell biologists in any field. An historical background and review of the relevant literature is included so that the reader will be fully prepared to use of modify the techniques and to troubleshoot results as required. All materials that are needed are thoroughly discussed so that any laboratory can adopt freeze substitution as a routine procedure.