31 research outputs found
Recommended from our members
Improving the statistical detection of regulated genes from microarray data using intensity-based variance estimation
BACKGROUND: Gene microarray technology provides the ability to study the regulation of thousands of genes simultaneously, but its potential is limited without an estimate of the statistical significance of the observed changes in gene expression. Due to the large number of genes being tested and the comparatively small number of array replicates (e.g., N = 3), standard statistical methods such as the Student's t-test fail to produce reliable results. Two other statistical approaches commonly used to improve significance estimates are a penalized t-test and a Z-test using intensity-dependent variance estimates. RESULTS: The performance of these approaches is compared using a dataset of 23 replicates, and a new implementation of the Z-test is introduced that pools together variance estimates of genes with similar minimum intensity. Significance estimates based on 3 replicate arrays are calculated using each statistical technique, and their accuracy is evaluated by comparing them to a reliable estimate based on the remaining 20 replicates. The reproducibility of each test statistic is evaluated by applying it to multiple, independent sets of 3 replicate arrays. Two implementations of a Z-test using intensity-dependent variance produce more reproducible results than two implementations of a penalized t-test. Furthermore, the minimum intensity-based Z-statistic demonstrates higher accuracy and higher or equal precision than all other statistical techniques tested. CONCLUSION: An intensity-based variance estimation technique provides one simple, effective approach that can improve p-value estimates for differentially regulated genes derived from replicated microarray datasets. Implementations of the Z-test algorithms are available at
Recommended from our members
Efficient In Silico Identification of a Common Insertion in the MAK Gene which Causes Retinitis Pigmentosa
Background: Next generation sequencing (NGS) offers a rapid and comprehensive method of screening for mutations associated with retinitis pigmentosa and related disorders. However, certain sequence alterations such as large insertions or deletions may remain undetected using standard NGS pipelines. One such mutation is a recently-identified Alu insertion into the Male Germ Cell-Associated Kinase (MAK) gene, which is missed by standard NGS-based variant callers. Here, we developed an in silico method of searching NGS raw sequence reads to detect this mutation, without the need to recalculate sequence alignments or to screen every sample by PCR. Methods: The Linux program grep was used to search for a 23 bp “probe” sequence containing the known junction sequence of the insert. A corresponding search was performed with the wildtype sequence. The matching reads were counted and further compared to the known sequences of the full wildtype and mutant genomic loci. (See https://github.com/MEEIBioinformaticsCenter/grepsearch.) Results: In a test sample set consisting of eleven previously published homozygous mutants, detection of the MAK-Alu insertion was validated with 100% sensitivity and specificity. As a discovery cohort, raw NGS reads from 1,847 samples (including custom and whole exome selective capture) were searched in ~1 hour on a local computer cluster, yielding an additional five samples with MAK-Alu insertions and solving two previously unsolved pedigrees. Of these, one patient was homozygous for the insertion, one compound heterozygous with a missense change on the other allele (c. 46G>A; p.Gly16Arg), and three were heterozygous carriers. Conclusions: Using the MAK-Alu grep program proved to be a rapid and effective method of finding a known, disease-causing Alu insertion in a large cohort of patients with NGS data. This simple approach avoids wet-lab assays or computationally expensive algorithms, and could also be used for other known disease-causing insertions and deletions
Unilateral Pigmentary Retinopathy Associated with Multiple Sclerosis
Unilateral retinitis pigmentosa (RP) is an incompletely characterized entity that can be mimicked by non-genetic disease processes including uveitis [1]. Although patients with uveitis have an approximately 10-fold elevated risk of developing multiple sclerosis (MS) [2], an association between MS and pigmentary retinopathy has not been described. Herein we report 5 cases of patients presenting with clinical diagnoses of unilateral RP and a history of MS
Argus—A New Database System for Web-Based Analysis of Multiple Microarray Data Sets
The ongoing revolution in microarray technology allows biologists studying gene expression to routinely collect >10(5) data points in a given experiment. Widely accessible and versatile database software is required to process this large amount of raw data into a format that facilitates the development of new biological insights. Here, we present a novel microarray database software system, named Argus, designed to process, analyze, manage, and publish microarray data. Argus imports the intensities and images of externally quantified microarray spots, performs normalization, and calculates ratios of gene expression between conditions. The database can be queried locally or over the Web, providing a convenient format for Web-publishing entire microarray data sets. Searches for regulated genes can be conducted across multiple experiments, and the integrated results incorporate images of the actual hybridization spots for artifact screening. Query results are presented in a clone- or gene-oriented fashion to rapidly identify highly regulated genes, and scatterplots of expression ratios allow an individual ratio to be interpreted in the context of all data points in the experiment. Algorithms were developed to optimize response times for queries of regulated genes. Supporting databases are updated easily to maintain current gene identity information, and hyperlinks to the Web provide access to descriptions of gene function. Query results also can be exported for higher-order analyses of expression patterns. This combination of features currently is not available in similar software. Argus is available at http://vessels.bwh.harvard.edu/software/Argus
Recommended from our members
Measuring ocular characteristics after gel injection adjustable keratoplasty (GIAK) in the rabbit
Gel Injection Adjustable Keratoplasty (GIAK) is a refractive surgery procedure which uses an ocular ring implant made of a polyethylene oxide hydrogel to cause a refractive change in the cornea. Unlike laser photo refractive keratectomy, GIAK does not interfere with the central cornea because the ring lies around the optical axis. Thus, vision can be assessed immediately after surgery. Our in vivo study was designed to quantify GIAK's effects on tissues, the biocompatibility of the polymer and in the process investigate which ocular changes in the rabbit model can be monitored with precision using current technology. Thirty-two young rabbits underwent a delamination in one eye, 22 of which were injected with a new polymeric gel. Corneal topography, keratometry, pachymetry, and tonometry were performed on both eyes for up to 105 days. All corneas flattened with growth. In GIAK animals, we found an average flattening of 6.51 +/- 1.23 diopters (p < 0.0001) relative to the fellow eye. No statistically significant regression over the 102 days was observed. Intraocular pressure dropped slightly by 0.69 +/- 1.21 mmHg (p equals 0.025), a clinically insignificant value, while no significant change was detected in corneal thickness. Keratometry can be tracked in rabbits after GIAK surgery from POD 1. Measuring unoperated fellow eyes allows for the effects of surgery to be assessed without bias from growth. Using this protocol, GIAK was shown to be stable. It was more difficult to draw conclusions from pachymetry, tonometry, and topography data
Tauroursodeoxycholic acid (TUDCA) protects photoreceptors from cell death after experimental retinal detachment.
Detachment of photoreceptors from the underlying retinal pigment epithelium is seen in various retinal disorders such as retinal detachment and age-related macular degeneration and leads to loss of photoreceptors and vision. Pharmacologic inhibition of photoreceptor cell death may prevent this outcome. This study tests whether systemic administration of tauroursodeoxycholic acid (TUDCA) can protect photoreceptors from cell death after experimental retinal detachment in rodents.Retinal detachment was created in rats by subretinal injection of hyaluronic acid. The animals were treated daily with vehicle or TUDCA (500 mg/kg). TUNEL staining was used to evaluate cell death. Photoreceptor loss was evaluated by measuring the relative thickness of the outer nuclear layer (ONL). Macrophage recruitment, oxidative stress, cytokine levels, and caspase levels were also quantified. Three days after detachment, TUDCA decreased the number of TUNEL-positive cells compared to vehicle (651±68/mm(2) vs. 1314±68/mm(2), P = 0.001) and prevented the reduction of ONL thickness ratio (0.84±0.03 vs. 0.65±0.03, P = 0.002). Similar results were obtained after 5 days of retinal detachment. Macrophage recruitment and expression levels of TNF-a and MCP-1 after retinal detachment were not affected by TUDCA treatment, whereas increases in activity of caspases 3 and 9 as well as carbonyl-protein adducts were almost completely inhibited by TUDCA treatment.Systemic administration of TUDCA preserved photoreceptors after retinal detachment, and was associated with decreased oxidative stress and caspase activity. TUDCA may be used as a novel therapeutic agent for preventing vision loss in diseases that are characterized by photoreceptor detachment
Recommended from our members
Laser scleral buckling: in-vitro quantification for Ho:YAG and Tm:YAG lasers
Pilot studies for laser scleral buckling made it clear that quantification of scleral shrinkage was required for precision and reproducibility of the treatment. For the quantification either the Ho:YAG (2.10 micrometers ) or the Tm:YAG (2.01 micrometers ) lasers were applied to the equatorial sclera of human cadaver eyes. Two slightly overlapping spots (2.8 mm (phi) ) were applied. Shrinkage rate was expressed as: [(Scleral length before treatment--Schleral length after treatment)/Schleral length before treatment] X 100(%). Shrinkage rate was measured changing several parameters. Total fluence, energy/pulse, scleral thickness, tissue temperature, age, and intraocular pressure. Shrinkage rate was found to be mainly function of total fluence attaining a maximum of 26 - 30% in adult and 46% in infant eyes at a 3 - 4 mm Hg intraocular pressure. Rising tissue temperature from room temperature to physiologic levels reduced the laser energy requirements but not the maximum shrinkage level. From the same shrinkage effect in the practical range of total fluence, less energy (56 - 60%) was required with the Tm:YAG laser. The data acquired in this study will help us construct an algorithm to predict the outcome of laser scleral buckling in patients
Characterization of the test sample set.
<p>A) Samples from previously reported patients with Alu insertion in <i>MAK</i> exon 9 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142614#pone.0142614.ref021" target="_blank">21</a>] and control samples were PCR amplified to detect homozygous alleles for Alu insertion and WT alleles. B) Sequence of the inserted element (280 bp Alu, 54 bp poly-A and 13 bp duplication of exon 9 sequence). C) Sanger sequence of the exon 9 Alu insertion breakpoints.</p