Large datasets of hundreds to thousands of individuals measuring RNA-seq in observational studies are becoming available. Many popular software packages for analysis of RNA-seq data were constructed to study differences in expression signatures in an experimental design with well-defined conditions (exposures). In contrast, observational studies may have varying levels of confounding transcript-exposure associations; further, exposure measures may vary from discrete (exposed, yes/no) to continuous (levels of exposure), with non-normal distributions of exposure. We compare popular software for gene expression-DESeq2, edgeR and limma-as well as linear regression-based analyses for studying the association of continuous exposures with RNA-seq. We developed a computation pipeline that includes transformation, filtering and generation of empirical null distribution of association P-values, and we apply the pipeline to compute empirical P-values with multiple testing correction. We employ a resampling approach that allows for assessment of false positive detection across methods, power comparison and the computation of quantile empirical P-values. The results suggest that linear regression methods are substantially faster with better control of false detections than other methods, even with the resampling method to compute empirical P-values. We provide the proposed pipeline with fast algorithms in an R package Olivia, and implemented it to study the associations of measures of sleep disordered breathing with RNA-seq in peripheral blood mononuclear cells in participants from the Multi-Ethnic Study of Atherosclerosis.

SUMMARY: Post-sequencing quality control is a crucial component of RNA sequencing (RNA-seq) data generation and analysis, as sample quality can be affected by sample storage, extraction and sequencing protocols. RNA-seq is increasingly applied to cohorts ranging from hundreds to tens of thousands of samples in size, but existing tools do not readily scale to these sizes, and were not designed for a wide range of sample types and qualities. Here, we describe RNA-SeQC 2, an efficient reimplementation of RNA-SeQC (DeLuca et al., 2012) that adds multiple metrics designed to characterize sample quality across a wide range of RNA-seq protocols. AVAILABILITY AND IMPLEMENTATION: The command-line tool, documentation and C++ source code are available at the GitHub repository https://github.com/getzlab/rnaseqc. Code and data for reproducing the figures in this paper are available at https://github.com/getzlab/rnaseqc2-paper. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Due to the limited depth of field of brightfield microscopes, it is usually impossible to image thick specimens entirely in focus. By optically sectioning the specimen, the in-focus information at the specimen's surface can be acquired over a range of images. Commonly based on a high-pass criterion, extended-depth-of-field methods aim at combining the in-focus information from these images into a single image of the texture on the specimen's surface. The topography provided by such methods is usually limited to a map of selected in-focus pixel positions and is inherently discretized along the axial direction, which limits its use for quantitative evaluation. In this paper, we propose a method that jointly estimates the texture and topography of a specimen from a series of brightfield optical sections; it is based on an image formation model that is described by the convolution of a thick specimen model with the microscope's point spread function. The problem is stated as a least-squares minimization where the texture and topography are updated alternately. This method also acts as a deconvolution when the in-focus PSF has a blurring effect, or when the true in-focus position falls in between two optical sections. Comparisons to state-of-the-art algorithms and experimental results demonstrate the potential of the proposed approach.

We present a framework for feature detection in 3-D using steerable filters. These filters can be designed to optimally respond to a particular type of feature by maximizing several Canny-like criteria. The detection process involves the analytical computation of the orientation and corresponding response of the template. A post-processing step consisting of the suppression of non-maximal values followed by thresholding to eliminate insignificant features concludes the detection procedure. We illustrate the approach with the design of feature templates for the detection of surfaces and curves, and demonstrate their efficiency with practical applications.