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 log2 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

TileProbe: modeling tiling array probe effects using publicly available data

Motivation: Individual probes on an Affymetrix tiling array usually behave differently. Modeling and removing these probe effects are critical for detecting signals from the array data. Current data processing techniques either require control samples or use probe sequences to model probe-specific variability, such as with MAT. Although the MAT approach can be applied without control samples, residual probe effects continue to distort the true biological signals

Soil carbon fluxes and stocks in a Great Lakes forest chronosequence

Author Posting. © The Authors, 2008. This is the author's version of the work. It is posted here by permission of Blackwell for personal use, not for redistribution. The definitive version was published in Global Change Biology 15 (2009): 145-155, doi:10.1111/j.1365-2486.2008.01741.x.We measured soil respiration and soil carbon stocks, as well as micrometeorological variables in a chronosequence of deciduous forests in Wisconsin and Michigan. The chronosequence consisted of (1) four recently disturbed stands, including a clearcut and repeatedly burned stand (burn), a blowdown and partial salvage stand (blowdown), a clearcut with sparse residual overstory (residual), and a regenerated stand from a complete clearcut (regenerated); (2) four young aspen (Populus tremuloides) stands in average age of 10 years; (3) four intermediate aspen stands in average age of 26 years; (4) four mature northern hardwood stands in average age of 73 years; and (5) an old-growth stand approximately 350 years old. We fitted site-based models and used continuous measurements of soil temperature to estimate cumulative soil respiration for the growing season of 2005 (days 133 to 295). Cumulative soil respiration in the growing season was estimated to be 513, 680, 747, 747, 794, 802, 690, and 571 gC m-2 in the burn, blowdown, residual, regenerated, young, intermediate, mature, and old-growth stands, respectively. The measured apparent temperature sensitivity of soil respiration was the highest in the regenerated stand, and declined from the young stands to the old-growth. Both cumulative soil respiration and basal soil respiration at 10˚C increased during stand establishment, peaked at intermediate age, and then decreased with age. Total soil carbon at 0-60 cm initially decreased after harvest, and increased after stands established. The old-growth stand accumulated carbon in deep layers of soils, but not in the surface soils. Our study suggests a complexity of long-term soil carbon dynamics, both in vertical depth and temporal scale.This work was primarily funded by the Office of Science/BER, U.S. Department of Energy Terrestrial Carbon Processes program (DE-FG02-00ER63023 and DE-FG02-03ER63682), and NASA Terrestrial Ecology Program (NNG05GD51G)

Are situation awareness and decision-making in driving totally conscious processes? Results of a Hazard Prediction task

Detecting danger in the driving environment is an indispensable task to guarantee safety which depends on the driver's ability to predict upcoming hazards. But does correct prediction lead to an appropriate response? This study advances hazard perception research by investigating the link between successful prediction and response selection. Three groups of drivers (learners, novices and experienced drivers) were recruited, with novice and experienced drivers further split into offender and non-offender groups. Specifically, this works aims to develop an improved Spanish Hazard Prediction Test and to explore the differences in Situation Awareness, (SA: perception, comprehension and prediction) and Decision-Making ("DM") among learners, younger inexperienced and experienced drivers and between driving offenders and non-offenders. The contribution of the current work is not only theoretical; the Hazard Prediction Test is also a valid way to test Hazard Perception. The test, as well as being useful as part of the test for a driving license, could also serve a purpose in the renewal of licenses after a ban or as a way of training drivers. A sample of 121 participants watched a series of driving video clips that ended with a sudden occlusion prior to a hazard. They then answered questions to assess their SA ("What is the hazard?" "Where is it located?" "What happens next?") and "DM" ("What would you do in this situation?"). This alternative to the Hazard Perception Test demonstrates a satisfactory internal consistency (Alpha=0.750), with eleven videos achieving discrimination indices above 0.30. Learners performed significantly worse than experienced drivers when required to identify and locate the hazard. Interestingly, drivers were more accurate in answering the "DM" question than questions regarding SA, suggesting that drivers can choose an appropriate response manoeuvre without a totally conscious knowledge of the exact hazard

MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors

BACKGROUND MicroRNA (miRNA) expression profiles have been described in pancreatic ductal adenocarcinoma (PDAC), but these have not been compared with pre-malignant pancreatic tumors. We wished to compare the miRNA expression signatures in pancreatic benign cystic tumors (BCT) of low and high malignant potential with PDAC, in order to identify miRNAs deregulated during PDAC development. The mechanistic consequences of miRNA dysregulation were further evaluated. METHODS Tissue samples were obtained at a tertiary pancreatic unit from individuals with BCT and PDAC. MiRNA profiling was performed using a custom microarray and results were validated using RT-qPCR prior to evaluation of miRNA targets. RESULTS Widespread miRNA down-regulation was observed in PDAC compared to low malignant potential BCT. We show that amongst those miRNAs down-regulated, miR-16, miR-126 and let-7d regulate known PDAC oncogenes (targeting BCL2, CRK and KRAS respectively). Notably, miR-126 also directly targets the KRAS transcript at a "seedless" binding site within its 3'UTR. In clinical specimens, miR-126 was strongly down-regulated in PDAC tissues, with an associated elevation in KRAS and CRK proteins. Furthermore, miR-21, a known oncogenic miRNA in pancreatic and other cancers, was not elevated in PDAC compared to serous microcystic adenoma (SMCA), but in both groups it was up-regulated compared to normal pancreas, implicating early up-regulation during malignant change. CONCLUSIONS Expression profiling revealed 21 miRNAs down-regulated in PDAC compared to SMCA, the most benign lesion that rarely progresses to invasive carcinoma. It appears that miR-21 up-regulation is an early event in the transformation from normal pancreatic tissue. MiRNA expression has the potential to distinguish PDAC from normal pancreas and BCT. Mechanistically the down-regulation of miR-16, miR-126 and let-7d promotes PDAC transformation by post-transcriptional up-regulation of crucial PDAC oncogenes. We show that miR-126 is able to directly target KRAS; re-expression has the potential as a therapeutic strategy against PDAC and other KRAS-driven cancers

Complement membrane attack and tumorigenesis: a systems biology approach

Tumor development driven by inflammation is now an established phenomenon, but the role that complement plays remains uncertain. Recent evidence has suggested that various components of the complement (C) cascade may influence tumor development in disparate ways; however, little attention has been paid to that of the membrane attack complex (MAC). This is despite abundant evidence documenting the effects of this complex on cell behavior, including cell activation, protection from/induction of apoptosis, release of inflammatory cytokines, growth factors, and ECM components and regulators, and the triggering of the NLRP3 inflammasome. Here we present a novel approach to this issue by using global gene expression studies in conjunction with a systems biology analysis. Using network analysis of MAC-responsive expression changes, we demonstrate a cluster of co-regulated genes known to have impact in the extracellular space and on the supporting stroma and with well characterized tumor-promoting roles. Network analysis highlighted the central role for EGF receptor activation in mediating the observed responses to MAC exposure. Overall, the study sheds light on the mechanisms by which sublytic MAC causes tumor cell responses and exposes a gene expression signature that implicates MAC as a driver of tumor progression. These findings have implications for understanding of the roles of complement and the MAC in tumor development and progression, which in turn will inform future therapeutic strategies in cancer

Hybridization modeling of oligonucleotide SNP arrays for accurate DNA copy number estimation

Affymetrix SNP arrays have been widely used for single-nucleotide polymorphism (SNP) genotype calling and DNA copy number variation inference. Although numerous methods have achieved high accuracy in these fields, most studies have paid little attention to the modeling of hybridization of probes to off-target allele sequences, which can affect the accuracy greatly. In this study, we address this issue and demonstrate that hybridization with mismatch nucleotides (HWMMN) occurs in all SNP probe-sets and has a critical effect on the estimation of allelic concentrations (ACs). We study sequence binding through binding free energy and then binding affinity, and develop a probe intensity composite representation (PICR) model. The PICR model allows the estimation of ACs at a given SNP through statistical regression. Furthermore, we demonstrate with cell-line data of known true copy numbers that the PICR model can achieve reasonable accuracy in copy number estimation at a single SNP locus, by using the ratio of the estimated AC of each sample to that of the reference sample, and can reveal subtle genotype structure of SNPs at abnormal loci. We also demonstrate with HapMap data that the PICR model yields accurate SNP genotype calls consistently across samples, laboratories and even across array platforms

Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice.

To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients. Top striatal modules implicated mHtt CAG length and age in graded impairment in the expression of identity genes for striatal medium spiny neurons and in dysregulation of cyclic AMP signaling, cell death and protocadherin genes. We used proteomics to confirm 790 genes and 5 striatal modules with CAG length-dependent dysregulation at the protein level, and validated 22 striatal module genes as modifiers of mHtt toxicities in vivo

A constrained polynomial regression procedure for estimating the local False Discovery Rate

<p>Abstract</p> <p>Background</p> <p>In the context of genomic association studies, for which a large number of statistical tests are performed simultaneously, the local False Discovery Rate (<it>lFDR</it>), which quantifies the evidence of a specific gene association with a clinical or biological variable of interest, is a relevant criterion for taking into account the multiple testing problem. The <it>lFDR </it>not only allows an inference to be made for each gene through its specific value, but also an estimate of Benjamini-Hochberg's False Discovery Rate (<it>FDR</it>) for subsets of genes.</p> <p>Results</p> <p>In the framework of estimating procedures without any distributional assumption under the alternative hypothesis, a new and efficient procedure for estimating the <it>lFDR </it>is described. The results of a simulation study indicated good performances for the proposed estimator in comparison to four published ones. The five different procedures were applied to real datasets.</p> <p>Conclusion</p> <p>A novel and efficient procedure for estimating <it>lFDR </it>was developed and evaluated.</p

Hearing effects from intermittent and continuous noise exposure in a study of Korean factory workers and firefighters

<p>Abstract</p> <p>Background</p> <p>South Korea and surrounding countries in East Asia are believed to have the highest proportion in the world of high frequency hearing loss due to occupational noise exposure, yet there has been limited information published in international journals, and limited information for control of noise in local workplaces beyond strategies from western countries. We exploit medical surveillance information from two worker groups to enhance local knowledge about noise-induced hearing loss and explore the possible importance of shift work to risk.</p> <p>Methods</p> <p>Four-years of hearing data were evaluated for 81 male farm machine factory workers and 371 male firefighters who had successfully completed a health examination and questionnaires for the duration of the study period. The averages of hearing thresholds at 2, 3, and 4 kHz were used as the primary end-point for comparison. Repeat measure analysis adjusted for age, exposure duration and smoking status was used to measure the difference in hearing threshold between the two groups.</p> <p>Results</p> <p>Noise levels were measured in the factory at a mean of 82 dBA, with a range of 66-97. No concurrent measurements were taken for the firefighters, but historic comparison values showed a wider range but a similar mean of 76-79 dBA. Although losses during follow-up were negligible, the factory workers had significantly (<it>P </it>< 0.0001) more hearing loss at the baseline of the study than the firefighters in both ears at 2, 3, and 4 kHz, adjusted for age, duration of employment and smoking status. Among those with 10 years of employment, mean losses at these frequencies among the factory workers fell into the impairment range (> 25 dB loss). Firefighters also showed increased losses associated with longer exposure duration, but these were significantly less marked. Losses at lower frequencies (< or = 1 kHz) were negligible in both groups.</p> <p>Conclusions</p> <p>Korean work environments with continuous noise exposure in the measured range should consider implementation of a hearing conservation program. Further evaluation of hearing loss in workers exposed to irregular or intermittent high noise levels, such as firefighters, is also warranted.</p