A verification protocol for the probe sequences of Affymetrix genome arrays reveals high probe accuracy for studies in mouse, human and rat

BACKGROUND: The Affymetrix GeneChip technology uses multiple probes per gene to measure its expression level. Individual probe signals can vary widely, which hampers proper interpretation. This variation can be caused by probes that do not properly match their target gene or that match multiple genes. To determine the accuracy of Affymetrix arrays, we developed an extensive verification protocol, for mouse arrays incorporating the NCBI RefSeq, NCBI UniGene Unique, NIA Mouse Gene Index, and UCSC mouse genome databases. RESULTS: Applying this protocol to Affymetrix Mouse Genome arrays (the earlier U74Av2 and the newer 430 2.0 array), the number of sequence-verified probes with perfect matches was no less than 85% and 95%, respectively; and for 74% and 85% of the probe sets all probes were sequence verified. The latter percentages increased to 80% and 94% after discarding one or two unverifiable probes per probe set, and even further to 84% and 97% when, in addition, allowing for one or two mismatches between probe and target gene. Similar results were obtained for other mouse arrays, as well as for human and rat arrays. Based on these data, refined chip definition files for all arrays are provided online. Researchers can choose the version appropriate for their study to (re)analyze expression data. CONCLUSION: The accuracy of Affymetrix probe sequences is higher than previously reported, particularly on newer arrays. Yet, refined probe set definitions have clear effects on the detection of differentially expressed genes. We demonstrate that the interpretation of the results of Affymetrix arrays is improved when the new chip definition files are used

One health: the importance of companion animal vector-borne diseases

The international prominence accorded the 'One Health' concept of co-ordinated activity of those involved in human and animal health is a modern incarnation of a long tradition of comparative medicine, with roots in the ancient civilizations and a golden era during the 19th century explosion of knowledge in the field of infectious disease research. Modern One Health tends to focus on zoonotic pathogens emerging from wildlife and production animal species, but one of the most significant One Health challenges is rabies for which there is a canine reservoir. This review considers the role of small companion animals in One Health and specifically addresses the major vector-borne infectious diseases that are shared by man, dogs and cats. The most significant of these are leishmaniosis, borreliosis, bartonellosis, ehrlichiosis, rickettsiosis and anaplasmosis. The challenges that lie ahead in this field of One Health are discussed, together with the role of the newly formed World Small Animal Veterinary Association One Health Committee

Gradual transition from mosaic to global DNA methylation patterns during deuterostome evolution

<p>Abstract</p> <p>Background</p> <p>DNA methylation by the Dnmt family occurs in vertebrates and invertebrates, including ascidians, and is thought to play important roles in gene regulation and genome stability, especially in vertebrates. However, the global methylation patterns of vertebrates and invertebrates are distinctive. Whereas almost all CpG sites are methylated in vertebrates, with the exception of those in CpG islands, the ascidian genome contains approximately equal amounts of methylated and unmethylated regions. Curiously, methylation status can be reliably estimated from the local frequency of CpG dinucleotides in the ascidian genome. Methylated and unmethylated regions tend to have few and many CpG sites, respectively, consistent with our knowledge of the methylation status of CpG islands and other regions in mammals. However, DNA methylation patterns and levels in vertebrates and invertebrates have not been analyzed in the same way.</p> <p>Results</p> <p>Using a new computational methodology based on the decomposition of the bimodal distributions of methylated and unmethylated regions, we estimated the extent of the global methylation patterns in a wide range of animals. We then examined the epigenetic changes <it>in silico </it>along the phylogenetic tree. We observed a gradual transition from fractional to global patterns of methylation in deuterostomes, rather than a clear demarcation between vertebrates and invertebrates. When we applied this methodology to six piscine genomes, some of which showed features similar to those of invertebrates.</p> <p>Conclusions</p> <p>The mammalian global DNA methylation pattern was probably not acquired at an early stage of vertebrate evolution, but gradually expanded from that of a more ancient organism.</p

Repetitive Elements May Comprise Over Two-Thirds of the Human Genome

Transposable elements (TEs) are conventionally identified in eukaryotic genomes by alignment to consensus element sequences. Using this approach, about half of the human genome has been previously identified as TEs and low-complexity repeats. We recently developed a highly sensitive alternative de novo strategy, P-clouds, that instead searches for clusters of high-abundance oligonucleotides that are related in sequence space (oligo “clouds”). We show here that P-clouds predicts >840 Mbp of additional repetitive sequences in the human genome, thus suggesting that 66%–69% of the human genome is repetitive or repeat-derived. To investigate this remarkable difference, we conducted detailed analyses of the ability of both P-clouds and a commonly used conventional approach, RepeatMasker (RM), to detect different sized fragments of the highly abundant human Alu and MIR SINEs. RM can have surprisingly low sensitivity for even moderately long fragments, in contrast to P-clouds, which has good sensitivity down to small fragment sizes (∼25 bp). Although short fragments have a high intrinsic probability of being false positives, we performed a probabilistic annotation that reflects this fact. We further developed “element-specific” P-clouds (ESPs) to identify novel Alu and MIR SINE elements, and using it we identified ∼100 Mb of previously unannotated human elements. ESP estimates of new MIR sequences are in good agreement with RM-based predictions of the amount that RM missed. These results highlight the need for combined, probabilistic genome annotation approaches and suggest that the human genome consists of substantially more repetitive sequence than previously believed

Identification of Maize Genes Associated with Host Plant Resistance or Susceptibility to Aspergillus flavus Infection and Aflatoxin Accumulation

infection and aflatoxin accumulation. inoculation were compared in two resistant maize inbred lines (Mp313E and Mp04∶86) in contrast to two susceptible maize inbred lines (Va35 and B73) by microarray analysis. Principal component analysis (PCA) was used to find genes contributing to the larger variances associated with the resistant or susceptible maize inbred lines. The significance levels of gene expression were determined by using SAS and LIMMA programs. Fifty candidate genes were selected and further investigated by quantitative RT-PCR (qRT-PCR) in a time-course study on Mp313E and Va35. Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E. infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation

Assignment of chromosomal locations for unassigned SNPs/scaffolds based on pair-wise linkage disequilibrium estimates

<p>Abstract</p> <p>Background</p> <p>Recent developments of high-density SNP chips across a number of species require accurate genetic maps. Despite rapid advances in genome sequence assembly and availability of a number of tools for creating genetic maps, the exact genome location for a number of SNPs from these SNP chips still remains unknown. We have developed a locus ordering procedure based on linkage disequilibrium (LODE) which provides estimation of the chromosomal positions of unaligned SNPs and scaffolds. It also provides an alternative means for verification of genetic maps. We exemplified LODE in cattle.</p> <p>Results</p> <p>The utility of the LODE procedure was demonstrated using data from 1,943 bulls genotyped for 73,569 SNPs across three different SNP chips. First, the utility of the procedure was tested by analysing the masked positions of 1,500 randomly-chosen SNPs with known locations (50 from each chromosome), representing three classes of minor allele frequencies (MAF), namely >0.05, 0.01<MAF ≤ 0.05 and 0.001<MAF ≤ 0.01. The efficiency (percentage of masked SNPs that could be assigned a location) was 96.7%, 30.6% and 2.0%; with an accuracy (the percentage of SNPs assigned correctly) of 99.9%, 98.9% and 33.3% in the three classes of MAF, respectively. The average precision for placement of the SNPs was 914, 3,137 and 6,853 kb, respectively. Secondly, 4,688 of 5,314 SNPs unpositioned in the Btau4.0 assembly were positioned using the LODE procedure. Based on these results, the positions of 485 unordered scaffolds were determined. The procedure was also used to validate the genome positions of 53,068 SNPs placed on Btau4.0 bovine assembly, resulting in identification of problem areas in the assembly. Finally, the accuracy of the LODE procedure was independently validated by comparative mapping on the hg18 human assembly.</p> <p>Conclusion</p> <p>The LODE procedure described in this study is an efficient and accurate method for positioning SNPs (MAF>0.05), for validating and checking the quality of a genome assembly, and offers a means for positioning of unordered scaffolds containing SNPs. The LODE procedure will be helpful in refining genome sequence assemblies, especially those being created from next-generation sequencing where high-throughput SNP discovery and genotyping platforms are integrated components of genome analysis.</p

Dr. PIAS: an integrative system for assessing the druggability of protein-protein interactions

<p>Abstract</p> <p>Background</p> <p>The amount of data on protein-protein interactions (PPIs) available in public databases and in the literature has rapidly expanded in recent years. PPI data can provide useful information for researchers in pharmacology and medicine as well as those in interactome studies. There is urgent need for a novel methodology or software allowing the efficient utilization of PPI data in pharmacology and medicine.</p> <p>Results</p> <p>To address this need, we have developed the 'Druggable Protein-protein Interaction Assessment System' (Dr. PIAS). Dr. PIAS has a meta-database that stores various types of information (tertiary structures, drugs/chemicals, and biological functions associated with PPIs) retrieved from public sources. By integrating this information, Dr. PIAS assesses whether a PPI is druggable as a target for small chemical ligands by using a supervised machine-learning method, support vector machine (SVM). Dr. PIAS holds not only known druggable PPIs but also all PPIs of human, mouse, rat, and human immunodeficiency virus (HIV) proteins identified to date.</p> <p>Conclusions</p> <p>The design concept of Dr. PIAS is distinct from other published PPI databases in that it focuses on selecting the PPIs most likely to make good drug targets, rather than merely collecting PPI data.</p

A novel transport mechanism for MOMP in Chlamydophila pneumoniae and its putative role in immune-therapy

Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE−/− mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane β-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events