Error, reproducibility and sensitivity: a pipeline for data processing of Agilent oligonucleotide expression arrays

Background: Expression microarrays are increasingly used to obtain large scale transcriptomic information on a wide range of biological samples. Nevertheless, there is still much debate on the best ways to process data, to design experiments and analyse the output. Furthermore, many of the more sophisticated mathematical approaches to data analysis in the literature remain inaccessible to much of the biological research community. In this study we examine ways of extracting and analysing a large data set obtained using the Agilent long oligonucleotide transcriptomics platform, applied to a set of human macrophage and dendritic cell samples. Results: We describe and validate a series of data extraction, transformation and normalisation steps which are implemented via a new R function. Analysis of replicate normalised reference data demonstrate that intrarray variability is small (only around 2 of the mean log signal), while interarray variability from replicate array measurements has a standard deviation (SD) of around 0.5 log(2) units (6 of mean). The common practise of working with ratios of Cy5/Cy3 signal offers little further improvement in terms of reducing error. Comparison to expression data obtained using Arabidopsis samples demonstrates that the large number of genes in each sample showing a low level of transcription reflect the real complexity of the cellular transcriptome. Multidimensional scaling is used to show that the processed data identifies an underlying structure which reflect some of the key biological variables which define the data set. This structure is robust, allowing reliable comparison of samples collected over a number of years and collected by a variety of operators. Conclusions: This study outlines a robust and easily implemented pipeline for extracting, transforming normalising and visualising transcriptomic array data from Agilent expression platform. The analysis is used to obtain quantitative estimates of the SD arising from experimental (non biological) intra- and interarray variability, and for a lower threshold for determining whether an individual gene is expressed. The study provides a reliable basis for further more extensive studies of the systems biology of eukaryotic cells.

On the generalized eigenvalue problem for the Rossby wave vertical velocity in the presence of mean flow and topography

In a series of papers, Killworth and Blundell have proposed to study the effects of a background mean flow and topography on Rossby wave propagation by means of a generalized eigenvalue problem formulated in terms of the vertical velocity, obtained from a linearization of the primitive equations of motion. However, it has been known for a number of years that this eigenvalue problem contains an error, which Killworth was prevented from correcting himself by his unfortunate passing and whose correction is therefore taken up in this note. Here, the author shows in the context of quasigeostrophic (QG) theory that the error can ulti- mately be traced to the fact that the eigenvalue problem for the vertical velocity is fundamentally a non- linear one (the eigenvalue appears both in the numerator and denominator), unlike that for the pressure. The reason that this nonlinear term is lacking in the Killworth and Blundell theory comes from neglecting the depth dependence of a depth-dependent term. This nonlinear term is shown on idealized examples to alter significantly the Rossby wave dispersion relation in the high-wavenumber regime but is otherwise irrelevant in the long-wave limit, in which case the eigenvalue problems for the vertical velocity and pressure are both linear. In the general dispersive case, however, one should first solve the generalized eigenvalue problem for the pressure vertical structure and, if needed, diagnose the vertical velocity vertical structure from the latter.

Systematic model forecast error in Rossby wave structure

Diabatic processes can alter Rossby wave structure; consequently errors arising from model processes propagate downstream. However, the chaotic spread of forecasts from initial condition uncertainty renders it difficult to trace back from root mean square forecast errors to model errors. Here diagnostics unaffected by phase errors are used, enabling investigation of systematic errors in Rossby waves in winter-season forecasts from three operational centers. Tropopause sharpness adjacent to ridges decreases with forecast lead time. It depends strongly on model resolution, even though models are examined on a common grid. Rossby wave amplitude reduces with lead time up to about five days, consistent with under-representation of diabatic modification and transport of air from the lower troposphere into upper-tropospheric ridges, and with too weak humidity gradients across the tropopause. However, amplitude also decreases when resolution is decreased. Further work is necessary to isolate the contribution from errors in the representation of diabatic processes.

How well can we measure the ocean’s mean dynamic topography from space?

Recent gravity missions have produced a dramatic improvement in our ability to measure the ocean’s mean dynamic topography (MDT) from space. To fully exploit this oceanic observation, however, we must quantify its error. To establish a baseline, we first assess the error budget for an MDT calculated using a 3rd generation GOCE geoid and the CLS01 mean sea surface (MSS). With these products, we can resolve MDT spatial scales down to 250 km with an accuracy of 1.7 cm, with the MSS and geoid making similar contributions to the total error. For spatial scales within the range 133–250 km the error is 3.0 cm, with the geoid making the greatest contribution. For the smallest resolvable spatial scales (80–133 km) the total error is 16.4 cm, with geoid error accounting for almost all of this. Relative to this baseline, the most recent versions of the geoid and MSS fields reduce the long and short-wavelength errors by 0.9 and 3.2 cm, respectively, but they have little impact in the medium-wavelength band. The newer MSS is responsible for most of the long-wavelength improvement, while for the short-wavelength component it is the geoid. We find that while the formal geoid errors have reasonable global mean values they fail capture the regional variations in error magnitude, which depend on the steepness of the sea floor topography.

An Ensemble Smoother with Error Estimates

A smoother introduced earlier by van Leeuwen and Evensen is applied to a problem in which real obser vations are used in an area with strongly nonlinear dynamics. The derivation is new , but it resembles an earlier derivation by van Leeuwen and Evensen. Again a Bayesian view is taken in which the prior probability density of the model and the probability density of the obser vations are combined to for m a posterior density . The mean and the covariance of this density give the variance-minimizing model evolution and its errors. The assumption is made that the prior probability density is a Gaussian, leading to a linear update equation. Critical evaluation shows when the assumption is justified. This also sheds light on why Kalman filters, in which the same ap- proximation is made, work for nonlinear models. By reference to the derivation, the impact of model and obser vational biases on the equations is discussed, and it is shown that Bayes’ s for mulation can still be used. A practical advantage of the ensemble smoother is that no adjoint equations have to be integrated and that error estimates are easily obtained. The present application shows that for process studies a smoother will give superior results compared to a filter , not only owing to the smooth transitions at obser vation points, but also because the origin of features can be followed back in time. Also its preference over a strong-constraint method is highlighted. Further more, it is argued that the proposed smoother is more efficient than gradient descent methods or than the representer method when error estimates are taken into account