Overestimation of alternative splicing caused by variable probe characteristics in exon arrays

In higher eukaryotes, alternative splicing is a common mechanism for increasing transcriptome diversity. Affymetrix exon arrays were designed as a tool for monitoring the relative expression levels of hundreds of thousands of known and predicted exons with a view to detecting alternative splicing events. In this article, we have analyzed exon array data from many different human and mouse tissues and have uncovered a systematic relationship between transcript-fold change and alternative splicing as reported by the splicing index. Evidence from dilution experiments and deep sequencing suggest that this effect is of technical rather than biological origin and that it is driven by sequence features of the probes. This effect is substantial and results in a 12-fold overestimation of alternative splicing events in genes that are differentially expressed. By cross-species exon array comparison, we could further show that the systematic bias persists even across species boundaries. Failure to consider this effect in data analysis would result in the reproducible false detection of apparently conserved alternative splicing events. Finally, we have developed a software in R called COSIE (Corrected Splicing Indices for Exon arrays) that for any given set of new exon array experiments corrects for the observed bias and improves the detection of alternative splicing (available at www.fmi.ch/groups/gbioinfo

N2O, NO, N2, and CO2 emissions from tropical savanna and grassland of Northern Australia: an incubation experiment with intact soil cores

Strong seasonal variability of hygric and thermal soil conditions are a defining environmental feature in northern Australia. However, how such changes affect the soil-atmosphere exchange of nitrous oxide (N2O), nitric oxide (NO) and dinitrogen (N2) is still not well explored. By incubating intact soil cores from four sites (three savanna, one pasture) under controlled soil temperatures (ST) and soil moisture (SM) we investigated the release of the trace gas fluxes of N2O, NO and carbon dioxide (CO2). Furthermore, the release of N2 due to denitrification was measured using the helium gas flow soil core technique. Under dry pre-incubation conditions NO and N2O emissions were very low (<7.0 ± 5.0 müg NO-N m-2 h-1; <0.0 ± 1.4 müg N2O-N m-2 h-1) or in the case of N2O, even a net soil uptake was observed. Substantial NO (max: 306.5 müg N m-2 h-1) and relatively small N2O pulse emissions (max: 5.8 ± 5.0 &müg N m-2 h-1) were recorded following soil wetting, but these pulses were short lived, lasting only up to 3 days. The total atmospheric loss of nitrogen was generally dominated by N2 emissions (82.4-99.3% of total N lost), although NO emissions contributed almost 43.2% to the total atmospheric nitrogen loss at 50% SM and 30 °C ST incubation settings (the contribution of N2 at these soil conditions was only 53.2%). N2O emissions were systematically higher for 3 of 12 sample locations, which indicates substantial spatial variability at site level, but on average soils acted as weak N2O sources or even sinks. By using a conservative upscale approach we estimate total annual emissions from savanna soils to average 0.12 kg N ha-1 yr-1 (N2O), 0.68 kg N ha-1 yr-1 (NO) and 6.65 kg N ha-1 yr-1 (N2). The analysis of long-term SM and ST records makes it clear that extreme soil saturation that can lead to high N2O and N2 emissions only occurs a few days per year and thus has little impact on the annual total. The potential contribution of nitrogen released due to pulse events compared to the total annual emissions was found to be of importance for NO emissions (contribution to total: 5-22%), but not for N2O emissions. Our results indicate that the total gaseous release of nitrogen from these soils is low and clearly dominated by loss in the form of inert nitrogen. Effects of seasonally varying soil temperature and moisture were detected, but were found to be low due to the small amounts of available nitrogen in the soils (total nitrogen <0.1%)

Overestimation of alternative splicing caused by variable probe characteristics in exon arrays

In higher eukaryotes, alternative splicing is a common mechanism for increasing transcriptome diversity. Affymetrix exon arrays were designed as a tool for monitoring the relative expression levels of hundreds of thousands of known and predicted exons with a view to detecting alternative splicing events. In this article, we have analyzed exon array data from many different human and mouse tissues and have uncovered a systematic relationship between transcript-fold change and alternative splicing as reported by the splicing index. Evidence from dilution experiments and deep sequencing suggest that this effect is of technical rather than biological origin and that it is driven by sequence features of the probes. This effect is substantial and results in a 12-fold overestimation of alternative splicing events in genes that are differentially expressed. By cross-species exon array comparison, we could further show that the systematic bias persists even across species boundaries. Failure to consider this effect in data analysis would result in the reproducible false detection of apparently conserved alternative splicing events. Finally, we have developed a software in R called COSIE (Corrected Splicing Indices for Exon arrays) that for any given set of new exon array experiments corrects for the observed bias and improves the detection of alternative splicing (available at www.fmi.ch/groups/gbioinfo)