iQuantitator: A tool for protein expression inference using iTRAQ

<p>Abstract</p> <p>Background</p> <p>Isobaric Tags for Relative and Absolute Quantitation (iTRAQ™) [Applied Biosystems] have seen increased application in differential protein expression analysis. To facilitate the growing need to analyze iTRAQ data, especially for cases involving multiple iTRAQ experiments, we have developed a modeling approach, statistical methods, and tools for estimating the relative changes in protein expression under various treatments and experimental conditions.</p> <p>Results</p> <p>This modeling approach provides a unified analysis of data from multiple iTRAQ experiments and links the observed quantity (reporter ion peak area) to the experiment design and the calculated quantity of interest (treatment-dependent protein and peptide fold change) through an additive model under log transformation. Others have demonstrated, through a case study, this modeling approach and noted the computational challenges of parameter inference in the unbalanced data set typical of multiple iTRAQ experiments. Here we present the development of an inference approach, based on hierarchical regression with batching of regression coefficients and Markov Chain Monte Carlo (MCMC) methods that overcomes some of these challenges. In addition to our discussion of the underlying method, we also present our implementation of the software, simulation results, experimental results, and sample output from the resulting analysis report.</p> <p>Conclusion</p> <p>iQuantitator's process-based modeling approach overcomes limitations in current methods and allows for application in a variety of experimental designs. Additionally, hypertext-linked documents produced by the tool aid in the interpretation and exploration of results.</p

Transcriptome Analysis and SNP Development Can Resolve Population Differentiation of Streblospio benedicti, a Developmentally Dimorphic Marine Annelid

Next-generation sequencing technology is now frequently being used to develop genomic tools for non-model organisms, which are generally important for advancing studies of evolutionary ecology. One such species, the marine annelid Streblospio benedicti, is an ideal system to study the evolutionary consequences of larval life history mode because the species displays a rare offspring dimorphism termed poecilogony, where females can produce either many small offspring or a few large ones. To further develop S. benedicti as a model system for studies of life history evolution, we apply 454 sequencing to characterize the transcriptome for embryos, larvae, and juveniles of this species, for which no genomic resources are currently available. Here we performed a de novo alignment of 336,715 reads generated by a quarter GS-FLX (Roche 454) run, which produced 7,222 contigs. We developed a novel approach for evaluating the site frequency spectrum across the transcriptome to identify potential signatures of selection. We also developed 84 novel single nucleotide polymorphism (SNP) markers for this species that are used to distinguish coastal populations of S. benedicti. We validated the SNPs by genotyping individuals of different developmental modes using the BeadXPress Golden Gate assay (Illumina). This allowed us to evaluate markers that may be associated with life-history mode

The Evolutionary Genetics and Emergence of Avian Influenza Viruses in Wild Birds

We surveyed the genetic diversity among avian influenza virus (AIV) in wild birds, comprising 167 complete viral genomes from 14 bird species sampled in four locations across the United States. These isolates represented 29 type A influenza virus hemagglutinin (HA) and neuraminidase (NA) subtype combinations, with up to 26% of isolates showing evidence of mixed subtype infection. Through a phylogenetic analysis of the largest data set of AIV genomes compiled to date, we were able to document a remarkably high rate of genome reassortment, with no clear pattern of gene segment association and occasional inter-hemisphere gene segment migration and reassortment. From this, we propose that AIV in wild birds forms transient “genome constellations,” continually reshuffled by reassortment, in contrast to the spread of a limited number of stable genome constellations that characterizes the evolution of mammalian-adapted influenza A viruses

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Imbalanced Expression of <i>Vcan</i> mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

<div><p>The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the <i>Vcan</i> null mouse called heart defect (<i>hdf</i>). Total absence of the <i>Vcan</i> gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, <i>Vcan</i>^(tm1Zim), of heart defects that results from deletion of exon 7 in the <i>Vcan</i> gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the <i>Vcan</i> gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of <i>Vcan</i> exon 7. The <i>Vcan</i>^(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.</p></div

Three-dimensional reconstruction and quantitative comparisons of wild-type and <i>Vcan</i> exon 7 cardiac OT associated cushions in E13.5 pc hearts.

<p>Significant differences were found in the volume of the pulmonary lumen and size of the pulmonary cushions in the <i>Vcan</i>^(tm1Zim) compared to wild-type littermates. The differences, visually apparent in the 3-dimensional comparisons (panel A; wild-type and <i>Vcan</i>^(tm1Zim)), were quantified using the AMIRA imaging software to measure the volumes of the cushion primordia of the pulmonary and aortic valves and of the pulmonary (brown) and aortic (purple) lumens. A significant increase (51%; *P value 0.047 panel D) was found in the size of the pulmonary cushions in the <i>Vcan</i>^(tm1Zim) hearts compared to wild-type littermates (panels D). The pulmonary lumens of the <i>Vcan</i>^(tm1Zim) cardiac outlets were found to be significantly (panel E; *P value 0.028 ) smaller (57%) then that of wild-type littermate controls. The aortic lumen volumes measured smaller but did not show a significant change (panel C). Brown-pulmonary lumen (PL); purple-aortic lumen (Ao); green-pulmonary cushions; yellow-aortic cushions.</p

Epithelial-mesenchymal 3D gel assay.

<p>Atrioventricular cushion explants from E10.5 pc hearts cultured on 3D hydrated collagen gels in the EMT assay produced significantly (p<0.05) less numbers of mesenchymal cells found below the surface of the gel from explants of <i>Vcan</i>^(tm1Zim) mice (average 37/explant; n = 3) vs. wild-type littermate controls (average 55/explant; n = 3).</p