BMC Genomics

DNA methylation patterns provide insight into epigenetic regulation in the Pacific oyster (Crassostrea gigas).

Publication Date: 2010 Aug 27 PMID: 20799955
Authors: Gavery, M. R. - Roberts, S. B.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: DNA methylation is an epigenetic mechanism with important regulatory functions in animals. While the mechanism itself is evolutionarily ancient, the distribution and function of DNA methylation is diverse both within and among phylogenetic groups. Although DNA methylation has been well studied in mammals, there are limited data on invertebrates, particularly molluscs. Here we characterize the distribution and investigate potential functions of DNA methylation in the Pacific oyster (Crassostrea gigas). RESULTS: Methylation sensitive PCR and bisulfite sequencing PCR approaches were used to identify CpG methylation in C.gigas genes and demonstrated that this species possesses intragenic methylation. In silico analysis of CpGo/e ratios in publicly available sequence data suggests that DNA methylation is a common feature of the C.gigas genome, and that specific functional categories of genes have significantly different levels of methylation. CONCLUSIONS: The Pacific oyster genome displays intragenic DNA methylation and contains genes necessary for DNA methylation in animals. Results of this investigation suggest that DNA methylation has regulatory functions in Crassostrea gigas, particularly in gene families that have inducible expression, including those involved in stress and environmental responses.

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Enumerating the gene sets in breast cancer, a "direct" alternative to hierarchical clustering.

Publication Date: 2010 Aug 23 PMID: 20731868
Authors: Mefford, D. - Mefford, J. A.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Two-way hierarchical clustering, with results visualized as heatmaps, has served as the method of choice for exploring structure in large matrices of expression data since the advent of microarrays. While it has delivered important insights, including a typology of breast cancer subtypes, it suffers from instability in the face of gene or sample selection, and an inability to detect small sets due to the effects of dominating sets of genes such as the estrogen-related genes in breast cancer. The rank-based partitioning algorithm introduced in this paper overcomes these limitations. It delivers results comparable to two-way hierarchical clustering, and much more. Applied systematically across a range of parameter settings, it enumerates a complete collection of the gene sets in a matrix of expression values. RESULTS: Applied to four large breast cancer datasets, this alternative method detects more than thirty sets of co-regulated genes, many of which are conserved across experiments and across platforms. Most of these sets are readily identified in biological terms, e.g., "estrogen", "erbb2", and 8p11-12, and several are clinically significant as prognostic of either increased survival ("adipose", "stromal"...) or the opposite ("proliferation", "immune/interferon", "histone",...). Of special interest are the sets that effectively factor "immune response" and "stromal signalling". CONCLUSION: The gene sets induced by the enumeration include many of the sets reported in the literature. In this regard these inventories confirm and consolidate findings from microarray-based work on breast cancer over the last decade. The enumerations also identify gene sets that have not been studied as of yet, some of which are prognostic of survival. The gene sets induced are robust, biologically meaningful, and, taken collectively, reveal a finer structure in existing breast cancer microarrays.

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DNA methylation status of nuclear-encoded mitochondrial genes underlies the tissue-dependent mitochondrial functions.

Publication Date: 2010 Aug 19 PMID: 20723256
Authors: Takasugi, M. - Yagi, S. - Hirabayashi, K. - Shiota, K.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Mitochondria are semi-autonomous, semi-self-replicating organelles harboring their own DNA (mitochondrial DNA, mtDNA), and their dysregulation is involved in the development of various diseases. While mtDNA does not generally undergo epigenetic modifications, almost all mitochondrial proteins are encoded by nuclear DNA. However, the epigenetic regulation of nuclear-encoded mitochondrial genes (nuclear mt genes) has not been comprehensively analyzed. RESULTS: We analyzed the DNA methylation status of 899 nuclear mt genes in the liver, brain, and heart tissues of mouse, and identified 636 nuclear mt genes carrying tissue-dependent and differentially methylated regions (T-DMRs). These nuclar mt genes are involved in various mitochondrial functions and they also include genes related to human diseases. T-DMRs regulate the expression of nuclear mt genes. Nuclear mt genes with tissue-specific hypomethylated T-DMRs were characterized by enrichment of the target genes of specific transcription factors such as FOXA2 in the liver, and CEBPA and STAT1 in the brain. CONCLUSIONS: A substantial proportion of nuclear mt genes contained T-DMRs, and the DNA methylation status of numerous T-DMRs should underlie tissue-dependent mitochondrial functions.

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Transcriptional profiling of root-knot nematode induced feeding sites in cowpea (Vigna unguiculata L. Walp.) using a soybean genome array.

Publication Date: 2010 PMID: 20723233
Authors: Das, S. - Ehlers, J. D. - Close, T. J. - Roberts, P. A.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The locus Rk confers resistance against several species of root-knot nematodes (Meloidogyne spp., RKN) in cowpea (Vigna unguiculata). Based on histological and reactive oxygen species (ROS) profiles, Rk confers a delayed but strong resistance mechanism without a hypersensitive reaction-mediated cell death process, which allows nematode development but blocks reproduction. RESULTS: Responses to M. incognita infection in roots of resistant genotype CB46 and a susceptible near-isogenic line (null-Rk) were investigated using a soybean Affymetrix GeneChip expression array at 3 and 9 days post-inoculation (dpi). At 9 dpi 552 genes were differentially expressed in incompatible interactions (infected resistant tissue compared with non-infected resistant tissue) and 1,060 genes were differentially expressed in compatible interactions (infected susceptible tissue compared with non-infected susceptible tissue). At 3 dpi the differentially expressed genes were 746 for the incompatible and 623 for the compatible interactions. When expression between infected resistant and susceptible genotypes was compared, 638 and 197 genes were differentially expressed at 9 and 3 dpi, respectively. CONCLUSIONS: In comparing the differentially expressed genes in response to nematode infection, a greater number and proportion of genes were down-regulated in the resistant than in the susceptible genotype, whereas more genes were up-regulated in the susceptible than in the resistant genotype. Gene ontology based functional categorization revealed that the typical defense response was partially suppressed in resistant roots, even at 9 dpi, allowing nematode juvenile development. Differences in ROS concentrations, induction of toxins and other defense related genes seem to play a role in this unique resistance mechanism.

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Transfer RNA gene arrangement and codon usage in vertebrate mitochondrial genomes: a new insight into gene order conservation.

Publication Date: 2010 Aug 19 PMID: 20723209
Authors: Satoh, T. P. - Sato, Y. - Masuyama, N. - Miya, M. - Nishida, M.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Mitochondrial (mt) gene arrangement has been highly conserved among vertebrates from jawless fishes to mammals for more than 500 million years. It remains unclear, however, whether such long-term persistence is a consequence of some constraints on the gene order. RESULTS: Based on the analysis of codon usage and tRNA gene locations, we suggest that tRNA gene order of the typical vertebrate mt-genomes may be important for their translational efficiency. The vertebrate mt-genome encodes 2 rRNA, 22 tRNA, and 13 transmembrane proteins consisting mainly of hydrophobic domains. We found that the tRNA genes specifying the hydrophobic residues were positioned close to the control region (CR), where the transcription efficiency is estimated to be relatively high. Using 47 vertebrate mt-genome sequences representing jawless fishes to mammals, we further found a correlation between codon usage and tRNA gene positions, implying that highly-used tRNA genes are located close to the CR. In addition, an analysis considering the asymmetric nature of mtDNA replication suggested that the tRNA loci that remain in single-strand for a longer time tend to have more guanine and thymine not suffering deamination mutations in their anticodon sites. CONCLUSIONS: Our analyses imply the existence of translational constraint acting on the vertebrate mt-gene arrangement. Such translational constraint, together with the deamination-related constraint, may have contributed to long-term maintenance of gene order.

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Combination of genomic approaches with functional genetic experiments reveals two modes of repression of yeast middle-phase meiosis genes.

Publication Date: 2010 Aug 17 PMID: 20716365
Authors: Klutstein, M. - Siegfried, Z. - Gispan, A. - Farkash-Amar, S. - Zinman, G. - Bar-Joseph, Z. - Simchen, G. - Simon, I.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Regulation of meiosis and sporulation in Saccharomyces cerevisiae is a model for a highly regulated developmental process. Meiosis middle phase transcriptional regulation is governed by two transcription factors: the activator Ndt80 and the repressor Sum1. It has been suggested that the competition between Ndt80 and Sum1 determines the temporal expression of their targets during middle meiosis. RESULTS: Using a combination of ChIP-on-chip and expression profiling, we characterized a middle phase transcriptional network and studied the relationship between Ndt80 and Sum1 during middle and late meiosis. While finding a group of genes regulated by both factors in a feed forward loop regulatory motif, our data also revealed a large group of genes regulated solely by Ndt80. Measuring the expression of all Ndt80 target genes in various genetic backgrounds (WT, sum1Delta and MK-ER-Ndt80 strains), allowed us to dissect the exact transcriptional network regulating each gene, which was frequently different than the one inferred from the binding data alone. CONCLUSION: These results highlight the need to perform detailed genetic experiments to determine the relative contribution of interactions in transcriptional regulatory networks.

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Multi-targeted priming for genome-wide gene expression assays.

Publication Date: 2010 Aug 17 PMID: 20716356
Authors: Adomas, A. B. - Lopez-Giraldez, F. - Clark, T. A. - Wang, Z. - Townsend, J. P.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. RESULTS: We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. CONCLUSIONS: Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed sequences within the genome.

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Gill transcriptome response to changes in environmental calcium in the green spotted puffer fish.

Publication Date: 2010 Aug 17 PMID: 20716350
Authors: Pinto, P. I. - Matsumura, H. - Thorne, M. A. - Power, D. M. - Terauchi, R. - Reinhardt, R. - Canario, A. V.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Calcium ion is tightly regulated in body fluids and for euryhaline fish, which are exposed to rapid changes in environmental [Ca2+], homeostasis is especially challenging. The gill is the main organ of active calcium uptake and therefore plays a crucial role in the maintenance of calcium ion homeostasis. To study the molecular basis of the short-term responses to changing calcium availability, the whole gill transcriptome obtained by Super Serial Analysis of Gene Expression (SuperSAGE) of the euryhaline teleost green spotted puffer fish, Tetraodon nigroviridis, exposed to water with altered [Ca2+] was analysed. RESULTS: Transfer of T. nigroviridis from 10 ppt water salinity containing 2.9 mM Ca2+ to high (10 mM Ca2+ ) and low (0.01mM Ca2+) calcium water of similar salinity for 2-12 h resulted in 1,339 differentially expressed SuperSAGE tags (26-bp transcript identifiers) in gills. Of these 869 tags (65%) were mapped to T. nigroviridis cDNAs or genomic DNA and 497 (57%) were assigned to known proteins. Thirteen percent of the genes matched multiple tags indicating alternative RNA transcripts. The main enriched gene ontology groups belong to Ca2+ signaling/homeostasis but also muscle contraction, cytoskeleton, energy production/homeostasis and tissue remodeling. K-means clustering identified co-expressed transcripts with distinct patterns in response to water [Ca2+] and exposure time. CONCLUSIONS: The generated transcript expression patterns provide a framework of novel water calcium-responsive genes in the gill during the initial response after transfer to different [Ca2+]. This molecular response entails initial perception of alterations, activation of signaling networks and effectors and suggests active remodeling of cytoskeletal proteins during the initial acclimation process. Genes related to energy production and energy homeostasis are also up-regulated, probably reflecting the increased energetic needs of the acclimation response. This study is the first genome-wide transcriptome analysis of fish gills and is an important resource for future research on the short-term mechanisms involved in the gill acclimation responses to environmental Ca2+ changes and osmoregulation.

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High-throughput SNP discovery and assay development in common bean.

Publication Date: 2010 PMID: 20712881
Authors: Hyten, D. L. - Song, Q. - Fickus, E. W. - Quigley, C. V. - Lim, J. S. - Choi, I. Y. - Hwang, E. Y. - Pastor-Corrales, M. - Cregan, P. B.
Journal: BMC Genomics

BACKGROUND: Next generation sequencing has significantly increased the speed at which single nucleotide polymorphisms (SNPs) can be discovered and subsequently used as molecular markers for research. Unfortunately, for species such as common bean (Phaseolus vulgaris L.) which do not have a whole genome sequence available, the use of next generation sequencing for SNP discovery is much more difficult and costly. To this end we developed a method which couples sequences obtained from the Roche 454-FLX system (454) with the Illumina Genome Analyzer (GA) for high-throughput SNP discovery. RESULTS: Using a multi-tier reduced representation library we discovered a total of 3,487 SNPs of which 2,795 contained sufficient flanking genomic sequence for SNP assay development. Using Sanger sequencing to determine the validation rate of these SNPs, we found that 86% are likely to be true SNPs. Furthermore, we designed a GoldenGate assay which contained 1,050 of the 3,487 predicted SNPs. A total of 827 of the 1,050 SNPs produced a working GoldenGate assay (79%). CONCLUSIONS: Through combining two next generation sequencing techniques we have developed a method that allows high-throughput SNP discovery in any diploid organism without the need of a whole genome sequence or the creation of normalized cDNA libraries. The need to only perform one 454 run and one GA sequencer run allows high-throughput SNP discovery with sufficient sequence for assay development to be performed in organisms, such as common bean, which have limited genomic resources.

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Modulation of the maternal immune system by the pre-implantation embryo.

Publication Date: 2010 PMID: 20707927
Authors: Walker, C. G. - Meier, S. - Littlejohn, M. D. - Lehnert, K. - Roche, J. R. - Mitchell, M. D.
Journal: BMC Genomics

BACKGROUND: A large proportion of pregnancy losses occur during the pre-implantation period, when the developing embryo is elongating rapidly and signalling its presence to the maternal system. The molecular mechanisms that prevent luteolysis and support embryo survival within the maternal environment are not well understood. To gain a more complete picture of these molecular events, genome-wide transcriptional profiles of reproductive day 17 endometrial tissue were determined in pregnant and cyclic Holstein-Friesian dairy cattle. RESULTS: Microarray analyses revealed 1,839 and 1,189 differentially expressed transcripts between pregnant and cyclic animals (with > or = 1.5 fold change in expression; P-value < 0.05, MTC Benjamini-Hochberg) in caruncular and intercaruncular endometrium respectively. Gene ontology and biological pathway analysis of differentially expressed genes revealed enrichment for genes involved in interferon signalling and modulation of the immune response in pregnant animals. CONCLUSION: The maternal immune system actively surveys the uterine environment during early pregnancy. The embryo modulates this response inducing the expression of endometrial molecules that suppress the immune response and promote maternal tolerance to the embryo. During this period of local immune suppression, genes of the innate immune response (in particular, antimicrobial genes) may function to protect the uterus against infection.

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Genetic validation of whole-transcriptome sequencing for mapping expression affected by cis-regulatory variation.

Publication Date: 2010 PMID: 20707912
Authors: Babak, T. - Garrett-Engele, P. - Armour, C. D. - Raymond, C. K. - Keller, M. P. - Chen, R. - Rohl, C. A. - Johnson, J. M. - Attie, A. D. - Fraser, H. B. - Schadt, E. E.
Journal: BMC Genomics

BACKGROUND: Identifying associations between genotypes and gene expression levels using microarrays has enabled systematic interrogation of regulatory variation underlying complex phenotypes. This approach has vast potential for functional characterization of disease states, but its prohibitive cost, given hundreds to thousands of individual samples from populations have to be genotyped and expression profiled, has limited its widespread application. RESULTS: Here we demonstrate that genomic regions with allele-specific expression (ASE) detected by sequencing cDNA are highly enriched for cis-acting expression quantitative trait loci (cis-eQTL) identified by profiling of 500 animals in parallel, with up to 90% agreement on the allele that is preferentially expressed. We also observed widespread noncoding and antisense ASE and identified several allele-specific alternative splicing variants. CONCLUSION: Monitoring ASE by sequencing cDNA from as little as one sample is a practical alternative to expression genetics for mapping cis-acting variation that regulates RNA transcription and processing.

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Deep sequencing-based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish.

Publication Date: 2010 PMID: 20707909
Authors: Xiang, L. X. - He, D. - Dong, W. R. - Zhang, Y. W. - Shao, J. Z.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Systematic research on fish immunogenetics is indispensable in understanding the origin and evolution of immune systems. This has long been a challenging task because of the limited number of deep sequencing technologies and genome backgrounds of non-model fish available. The newly developed Solexa/Illumina RNA-seq and Digital gene expression (DGE) are high-throughput sequencing approaches and are powerful tools for genomic studies at the transcriptome level. This study reports the transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus using RNA-seq and DGE in an attempt to gain insights into the immunogenetics of marine fish. RESULTS: RNA-seq analysis generated 169,950 non-redundant consensus sequences, among which 48,987 functional transcripts with complete or various length encoding regions were identified. More than 52% of these transcripts are possibly involved in approximately 219 known metabolic or signalling pathways, while 2,673 transcripts were associated with immune-relevant genes. In addition, approximately 8% of the transcripts appeared to be fish-specific genes that have never been described before. DGE analysis revealed that the host transcriptome profile of Vibrio harveyi-challenged L. japonicus is considerably altered, as indicated by the significant up- or down-regulation of 1,224 strong infection-responsive transcripts. Results indicated an overall conservation of the components and transcriptome alterations underlying innate and adaptive immunity in fish and other vertebrate models. Analysis suggested the acquisition of numerous fish-specific immune system components during early vertebrate evolution. CONCLUSION: This study provided a global survey of host defence gene activities against bacterial challenge in a non-model marine fish. Results can contribute to the in-depth study of candidate genes in marine fish immunity, and help improve current understanding of host-pathogen interactions and evolutionary history of immunogenetics from fish to mammals.

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Positional differences in the wound transcriptome of skin and oral mucosa.

Publication Date: 2010 Aug 12 PMID: 20704739
Authors: Chen, L. - Arbieva, Z. H. - Guo, S. - Marucha, P. T. - Mustoe, T. A. - Dipietro, L. A.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: When compared to skin, oral mucosal wounds heal rapidly and with reduced scar formation. Recent studies suggest that intrinsic differences in inflammation, growth factor production, levels of stem cells, and cellular proliferation capacity may underlie the exceptional healing that occurs in oral mucosa. The current study was designed to compare the transcriptomes of oral mucosal and skin wounds in order to identify critical differences in the healing response at these two sites using an unbiased approach. RESULTS: Using microarray analysis, we explored the differences in gene expression in skin and oral mucosal wound healing in a murine model of paired equivalent sized wounds. Samples were examined from days 0 to 10 and spanned all stages of the wound healing process. Using unwounded matched tissue as a control, filtering identified 1,479 probe sets in skin wounds yet only 502 probe sets in mucosal wounds that were significantly differentially expressed over time. Clusters of genes that showed similar patterns of expression were also identified in each wound type. Analysis of functionally related gene expression demonstrated dramatically different reactions to injury between skin and mucosal wounds. To explore whether site-specific differences might be derived from intrinsic differences in cellular responses at each site, we compared the response of isolated epithelial cells from skin and oral mucosa to a defined in vitro stimulus. When cytokine levels were measured, epithelial cells from skin produced significantly higher amounts of proinflammatory cytokines than cells from oral mucosa. CONCLUSIONS: The results provide the first detailed molecular profile of the site-specific differences in the genetic response to injury in mucosa and skin, and suggest the divergent reactions to injury may derive from intrinsic differences in the cellular responses at each site.

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The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology.

Publication Date: 2010 PMID: 20701780
Authors: Rajagopala, S. V. - Yamamoto, N. - Zweifel, A. E. - Nakamichi, T. - Huang, H. K. - Mendez-Rios, J. D. - Franca-Koh, J. - Boorgula, M. P. - Fujita, K. - Suzuki, K. - Hu, J. C. - Wanner, B. L. - Mori, H. - Uetz, P.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Systems biology and functional genomics require genome-wide datasets and resources. Complete sets of cloned open reading frames (ORFs) have been made for about a dozen bacterial species and allow researchers to express and study complete proteomes in a high-throughput fashion. RESULTS: We have constructed an open reading frame (ORFeome) collection of 3974 or 94% of the known Escherichia coli K-12 ORFs in Gateway(R) entry vector pENTR/Zeo. The collection has been used for protein expression and protein interaction studies. For example, we have compared interactions among YgjD, YjeE and YeaZ proteins in E. coli, Streptococcus pneumoniae, and Staphylococcus aureus. We also compare this ORFeome with other Gateway-compatible bacterial ORFeomes and show its utility for comparative functional genomics. CONCLUSIONS: The E. coli ORFeome provides a useful resource for functional genomics and other areas of protein research in a highly flexible format. Our comparison with other ORFeomes makes comparative analyses straighforward and facilitates direct comparisons of many proteins across many genomes.

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SNP discovery by high-throughput sequencing in soybean.

Publication Date: 2010 PMID: 20701770
Authors: Wu, X. - Ren, C. - Joshi, T. - Vuong, T. - Xu, D. - Nguyen, H. T.
Journal: BMC Genomics

BACKGROUND: With the advance of new massively parallel genotyping technologies, quantitative trait loci (QTL) fine mapping and map-based cloning become more achievable in identifying genes for important and complex traits. Development of high-density genetic markers in the QTL regions of specific mapping populations is essential for fine-mapping and map-based cloning of economically important genes. Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation existing between any diverse genotypes that are usually used for QTL mapping studies. The massively parallel sequencing technologies (Roche GS/454, Illumina GA/Solexa, and ABI/SOLiD), have been widely applied to identify genome-wide sequence variations. However, it is still remains unclear whether sequence data at a low sequencing depth are enough to detect the variations existing in any QTL regions of interest in a crop genome, and how to prepare sequencing samples for a complex genome such as soybean. Therefore, with the aims of identifying SNP markers in a cost effective way for fine-mapping several QTL regions, and testing the validation rate of the putative SNPs predicted with Solexa short sequence reads at a low sequencing depth, we evaluated a pooled DNA fragment reduced representation library and SNP detection methods applied to short read sequences generated by Solexa high-throughput sequencing technology. RESULTS: A total of 39,022 putative SNPs were identified by the Illumina/Solexa sequencing system using a reduced representation DNA library of two parental lines of a mapping population. The validation rates of these putative SNPs predicted with low and high stringency were 72% and 85%, respectively. One hundred sixty four SNP markers resulted from the validation of putative SNPs and have been selectively chosen to target a known QTL, thereby increasing the marker density of the targeted region to one marker per 42 K bp. CONCLUSIONS: We have demonstrated how to quickly identify large numbers of SNPs for fine mapping of QTL regions by applying massively parallel sequencing combined with genome complexity reduction techniques. This SNP discovery approach is more efficient for targeting multiple QTL regions in a same genetic population, which can be applied to other crops.

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Highly-multiplexed SNP genotyping for genetic mapping and germplasm diversity studies in pea.

Publication Date: 2010 Aug 11 PMID: 20701750
Authors: Deulvot, C. - Charrel, H. - Marty, A. - Jacquin, F. - Donnadieu, C. - Lejeune-Henaut, I. - Burstin, J. - Aubert, G.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Single Nucleotide Polymorphisms (SNPs) can be used as genetic markers for applications such as genetic diversity studies or genetic mapping. New technologies now allow genotyping hundreds to thousands of SNPs in a single reaction. In order to evaluate the potential of these technologies in pea, we selected a custom 384-SNP set using SNPs discovered in Pisum through the resequencing of gene fragments in different genotypes and by compiling genomic sequence data present in databases. We then designed an Illumina GoldenGate assay to genotype both a Pisum germplasm collection and a genetic mapping population with the SNP set. RESULTS: We obtained clear allelic data for more than 92% of the SNPs (356 out of 384). Interestingly, the technique was successful for all the genotypes present in the germplasm collection, including those from species or subspecies different from the P. sativum ssp sativum used to generate sequences. By genotyping the mapping population with the SNP set, we obtained a genetic map and map positions for 37 new gene markers. CONCLUSION: Our results show that the Illumina GoldenGate assay can be used successfully for high-throughput SNP genotyping of diverse germplasm in pea. This genotyping approach will simplify genotyping procedures for association mapping or diversity studies purposes and open new perspectives in legume genomics.

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A novel tissue-specific meta-analysis approach for gene expression predictions, initiated with a mammalian gene expression testis database.

Publication Date: 2010 PMID: 20699007
Authors: Acharya, K. K. - Chandrashekar, D. S. - Chitturi, N. - Shah, H. - Malhotra, V. - Sreelakshmi, K. S. - Deepti, H. - Bajpai, A. - Davuluri, S. - Bora, P. - Rao, L.
Journal: BMC Genomics

BACKGROUND: In the recent years, there has been a rise in gene expression profiling reports. Unfortunately, it has not been possible to make maximum use of available gene expression data. Many databases and programs can be used to derive the possible expression patterns of mammalian genes, based on existing data. However, these available resources have limitations. For example, it is not possible to obtain a list of genes that are expressed in certain conditions. To overcome such limitations, we have taken up a new strategy to predict gene expression patterns using available information, for one tissue at a time. RESULTS: The first step of this approach involved manual collection of maximum data derived from large-scale (genome-wide) gene expression studies, pertaining to mammalian testis. These data have been compiled into a Mammalian Gene Expression Testis-database (MGEx-Tdb). This process resulted in a richer collection of gene expression data compared to other databases/resources, for multiple testicular conditions. The gene-lists collected this way in turn were exploited to derive a 'consensus' expression status for each gene, across studies. The expression information obtained from the newly developed database mostly agreed with results from multiple small-scale studies on selected genes. A comparative analysis showed that MGEx-Tdb can retrieve the gene expression information more efficiently than other commonly used databases. It has the ability to provide a clear expression status (transcribed or dormant) for most genes, in the testis tissue, under several specific physiological/experimental conditions and/or cell-types. CONCLUSIONS: Manual compilation of gene expression data, which can be a painstaking process, followed by a consensus expression status determination for specific locations and conditions, can be a reliable way of making use of the existing data to predict gene expression patterns. MGEx-Tdb provides expression information for 14 different combinations of specific locations and conditions in humans (25,158 genes), 79 in mice (22,919 genes) and 23 in rats (14,108 genes). It is also the first system that can predict expression of genes with a 'reliability-score', which is calculated based on the extent of agreements and contradictions across gene-sets/studies. This new platform is publicly available at the following web address: http://resource.ibab.ac.in/MGEx-Tdb/.

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A complete DNA sequence map of the ovine Major Histocompatibility Complex.

Publication Date: 2010 PMID: 20698968
Authors: Gao, J. - Liu, K. - Liu, H. - Blair, H. T. - Li, G. - Chen, C. - Tan, P. - Ma, R. Z.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The ovine Major Histocompatibility Complex (MHC) harbors clusters of genes involved in overall resistance/susceptibility of an animal to infectious pathogens. However, only a limited number of ovine MHC genes have been identified and no adequate sequence information is available, as compared to those of swine and bovine. We previously constructed a BAC clone-based physical map that covers entire class I, class II and class III region of ovine MHC. Here we describe the assembling of a complete DNA sequence map for the ovine MHC by shotgun sequencing of 26 overlapping BAC clones. RESULTS: DNA shotgun sequencing generated approximately 8-fold genome equivalent data that were successfully assembled into a finished sequence map of the ovine MHC. The sequence map spans approximately 2,434,000 nucleotides in length, covering almost all of the MHC loci currently known in the sheep and cattle. Gene annotation resulted in the identification of 177 protein-coding genes/ORFs, among which 145 were not previously reported in the sheep, and 10 were ovine species specific, absent in cattle or other mammals. A comparative sequence analyses among human, sheep and cattle revealed a high conservation in the MHC structure and loci order except for the class II, which were divided into IIa and IIb subregions in the sheep and cattle, separated by a large piece of non-MHC autosome of approximately 18.5 Mb. In addition, a total of 18 non-protein-coding microRNAs were predicted in the ovine MHC region for the first time. CONCLUSION: An ovine MHC DNA sequence map was successfully assembled by shotgun sequencing of 26 overlapping BAC clone. This makes the sheep the second ruminant species for which the complete MHC sequence information is available for evolution and functional studies, following that of the bovine. The results of the comparative analysis support a hypothesis that an inversion of the ancestral chromosome containing the MHC has shaped the MHC structures of ruminants, as we currently observed in the sheep and cattle. Identification of relative large numbers of microRNAs in the ovine MHC region helps to provide evidence that microRNAs are actively involved in the regulation of MHC gene expression and function.

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Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans.

Publication Date: 2010 PMID: 20691096
Authors: Sha, K. - Gu, S. G. - Pantalena-Filho, L. C. - Goh, A. - Fleenor, J. - Blanchard, D. - Krishna, C. - Fire, A.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Tissue differentiation is accompanied by genome-wide changes in the underlying chromatin structure and dynamics, or epigenome. By controlling when, where, and what regulatory factors have access to the underlying genomic DNA, the epigenome influences the cell's transcriptome and ultimately its function. Existing genomic methods for analyzing cell-type-specific changes in chromatin generally involve two elements: (i) a source for purified cells (or nuclei) of distinct types, and (ii) a specific treatment that partitions or degrades chromatin by activity or structural features. For many cell types of great interest, such assays are limited by our inability to isolate the relevant cell populations in an organism or complex tissue containing an intertwined mixture of other cells. This limitation has confined available knowledge of chromatin dynamics to a narrow range of biological systems (cell types that can be sorted/separated/dissected in large numbers and tissue culture models) or to amalgamations of diverse cell types (tissue chunks, whole organisms). RESULTS: Transgene-driven expression of DNA/chromatin modifying enzymes provides one opportunity to query chromatin structures in expression-defined cell subsets. In this work we combine in vivo expression of a bacterial DNA adenine methyltransferase (DAM) with high throughput sequencing to sample tissue-specific chromatin accessibility on a genome-wide scale. We have applied the method (DALEC: Direct Asymmetric Ligation End Capture) towards mapping a cell-type-specific view of genome accessibility as a function of differentiated state. Taking advantage of C. elegans strains expressing the DAM enzyme in diverse tissues (body wall muscle, gut, and hypodermis), our efforts yield a genome-wide dataset measuring chromatin accessibility at each of 538,000 DAM target sites in the C. elegans (diploid) genome. CONCLUSIONS: Validating the DALEC mapping results, we observe a strong association between observed coverage by nucleosomes and low DAM accessibility. Strikingly, we observed no extended regions of inaccessible chromatin for any of the tissues examined. These results are consistent with "local choreography" models in which differential gene expression is driven by intricate local rearrangements of chromatin structure rather than gross impenetrability of large chromosomal regions.

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Analysis of intra-genomic GC content homogeneity within prokaryotes.

Publication Date: 2010 PMID: 20691090
Authors: Bohlin, J. - Snipen, L. - Hardy, S. P. - Kristoffersen, A. B. - Lagesen, K. - Donsvik, T. - Skjerve, E. - Ussery, D. W.
Journal: BMC Genomics

BACKGROUND: Bacterial genomes possess varying GC content (total guanines (Gs) and cytosines (Cs) per total of the four bases within the genome) but within a given genome, GC content can vary locally along the chromosome, with some regions significantly more or less GC rich than on average. We have examined how the GC content varies within microbial genomes to assess whether this property can be associated with certain biological functions related to the organism's environment and phylogeny. We utilize a new quantity GCVAR, the intra-genomic GC content variability with respect to the average GC content of the total genome. A low GCVAR indicates intra-genomic GC homogeneity and high GCVAR heterogeneity. RESULTS: The regression analyses indicated that GCVAR was significantly associated with domain (i.e. archaea or bacteria), phylum, and oxygen requirement. GCVAR was significantly higher among anaerobes than both aerobic and facultative microbes. Although an association has previously been found between mean genomic GC content and oxygen requirement, our analysis suggests that no such association exits when phylogenetic bias is accounted for. A significant association between GCVAR and mean GC content was also found but appears to be non-linear and varies greatly among phyla. CONCLUSIONS: Our findings show that GCVAR is linked with oxygen requirement, while mean genomic GC content is not. We therefore suggest that GCVAR should be used as a complement to mean GC content.

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Comparative analysis of expressed sequence tags from three castes and two life stages of the termite Reticulitermes flavipes.

Publication Date: 2010 PMID: 20691076
Authors: Steller, M. M. - Kambhampati, S. - Caragea, D.
Journal: BMC Genomics

BACKGROUND: Termites (Isoptera) are eusocial insects whose colonies consist of morphologically and behaviorally specialized castes of sterile workers and soldiers, and reproductive alates. Previous studies on eusocial insects have indicated that caste differentiation and behavior are underlain by differential gene expression. Although much is known about gene expression in the honey bee, Apis mellifera, termites remain relatively understudied in this regard. Therefore, our objective was to assemble an expressed sequence tag (EST) data base for the eastern subterranean termite, Reticulitermes flavipes, for future gene expression studies. RESULTS: Soldier, worker, and alate caste and two larval cDNA libraries were constructed, and approximately 15,000 randomly chosen clones were sequenced to compile an EST data base. Putative gene functions were assigned based on a BLASTX Swissprot search. Categorical in silico expression patterns for each library were compared using the R-statistic. A significant proportion of the ESTs of each caste and life stages had no significant similarity to those in existing data bases. All cDNA libraries, including those of non-reproductive worker and soldier castes, contained sequences with putative reproductive functions. Genes that showed a potential expression bias among castes included a putative antibacterial humoral response and translation elongation protein in soldiers and a chemosensory protein in alates. CONCLUSIONS: We have expanded upon the available sequences for R. flavipes and utilized an in silico method to compare gene expression in different castes of an eusocial insect. The in silico analysis allowed us to identify several genes which may be differentially expressed and involved in caste differences. These include a gene overrepresented in the alate cDNA library with a predicted function of neurotransmitter secretion or cholesterol absorption and a gene predicted to be involved in protein biosynthesis and ligase activity that was overrepresented in the late larval stage cDNA library. The EST data base and analyses reported here will be a valuable resource for future studies on the genomics of R. flavipes and other termites.

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Transcriptome analysis of the oil-rich seed of the bioenergy crop Jatropha curcas L.

Publication Date: 2010 PMID: 20691070
Authors: Costa, G. G. - Cardoso, K. C. - Del Bem, L. E. - Lima, A. C. - Cunha, M. A. - de Campos-Leite, L. - Vicentini, R. - Papes, F. - Moreira, R. C. - Yunes, J. A. - Campos, F. A. - Da Silva, M. J.
Journal: BMC Genomics

BACKGROUND: To date, oil-rich plants are the main source of biodiesel products. Because concerns have been voiced about the impact of oil-crop cultivation on the price of food commodities, the interest in oil plants not used for food production and amenable to cultivation on non-agricultural land has soared. As a non-food, drought-resistant and oil-rich crop, Jatropha curcas L. fulfils many of the requirements for biofuel production. RESULTS: We have generated 13,249 expressed sequence tags (ESTs) from developing and germinating Jatropha seeds. This strategy allowed us to detect most known genes related to lipid synthesis and degradation. We have also identified ESTs coding for proteins that may be involved in the toxicity of Jatropha seeds. Another unexpected finding is the high number of ESTs containing transposable element-related sequences in the developing seed library (800) when contrasted with those found in the germinating seed library (80). CONCLUSIONS: The sequences generated in this work represent a considerable increase in the number of sequences deposited in public databases. These results can be used to produce genetically improved varieties of Jatropha with increased oil yields, different oil compositions and better agronomic characteristics.

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MLTreeMap--accurate Maximum Likelihood placement of environmental DNA sequences into taxonomic and functional reference phylogenies.

Publication Date: 2010 PMID: 20687950
Authors: Stark, M. - Berger, S. A. - Stamatakis, A. - von Mering, C.
Journal: BMC Genomics

BACKGROUND: Shotgun sequencing of environmental DNA is an essential technique for characterizing uncultivated microbes in situ. However, the taxonomic and functional assignment of the obtained sequence fragments remains a pressing problem. RESULTS: Existing algorithms are largely optimized for speed and coverage; in contrast, we present here a software framework that focuses on a restricted set of informative gene families, using Maximum Likelihood to assign these with the best possible accuracy. This framework ('MLTreeMap'; http://mltreemap.org/) uses raw nucleotide sequences as input, and includes hand-curated, extensible reference information. CONCLUSIONS: We discuss how we validated our pipeline using complete genomes as well as simulated and actual environmental sequences.

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A proteogenomic update to Yersinia: enhancing genome annotation.

Publication Date: 2010 PMID: 20687929
Authors: Payne, S. H. - Huang, S. T. - Pieper, R.
Journal: BMC Genomics

BACKGROUND: Modern biomedical research depends on a complete and accurate proteome. With the widespread adoption of new sequencing technologies, genome sequences are generated at a near exponential rate, diminishing the time and effort that can be invested in genome annotation. The resulting gene set contains numerous errors in even the most basic form of annotation: the primary structure of the proteins. RESULTS: The application of experimental proteomics data to genome annotation, called proteogenomics, can quickly and efficiently discover misannotations, yielding a more accurate and complete genome annotation. We present a comprehensive proteogenomic analysis of the plague bacterium, Yersinia pestis KIM. We discover non-annotated genes, correct protein boundaries, remove spuriously annotated ORFs, and make major advances towards accurate identification of signal peptides. Finally, we apply our data to 21 other Yersinia genomes, correcting and enhancing their annotations. CONCLUSIONS: In total, 141 gene models were altered and have been updated in RefSeq and Genbank, which can be accessed seamlessly through any NCBI tool (e.g. blast) or downloaded directly. Along with the improved gene models we discover new, more accurate means of identifying signal peptides in proteomics data.

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Genome mapping and characterization of the Anopheles gambiae heterochromatin.

Publication Date: 2010 PMID: 20684766
Authors: Sharakhova, M. V. - George, P. - Brusentsova, I. V. - Leman, S. C. - Bailey, J. A. - Smith, C. D. - Sharakhov, I. V.
Journal: BMC Genomics

BACKGROUND: Heterochromatin plays an important role in chromosome function and gene regulation. Despite the availability of polytene chromosomes and genome sequence, the heterochromatin of the major malaria vector Anopheles gambiae has not been mapped and characterized. RESULTS: To determine the extent of heterochromatin within the An. gambiae genome, genes were physically mapped to the euchromatin-heterochromatin transition zone of polytene chromosomes. The study found that a minimum of 232 genes reside in 16.6 Mb of mapped heterochromatin. Gene ontology analysis revealed that heterochromatin is enriched in genes with DNA-binding and regulatory activities. Immunostaining of the An. gambiae chromosomes with antibodies against Drosophila melanogaster heterochromatin protein 1 (HP1) and the nuclear envelope protein lamin Dm0 identified the major invariable sites of the proteins' localization in all regions of pericentric heterochromatin, diffuse intercalary heterochromatin, and euchromatic region 9C of the 2R arm, but not in the compact intercalary heterochromatin. To better understand the molecular differences among chromatin types, novel Bayesian statistical models were developed to analyze genome features. The study found that heterochromatin and euchromatin differ in gene density and the coverage of retroelements and segmental duplications. The pericentric heterochromatin had the highest coverage of retroelements and tandem repeats, while intercalary heterochromatin was enriched with segmental duplications. We also provide evidence that the diffuse intercalary heterochromatin has a higher coverage of DNA transposable elements, minisatellites, and satellites than does the compact intercalary heterochromatin. The investigation of 42-Mb assembly of unmapped genomic scaffolds showed that it has molecular characteristics similar to cytologically mapped heterochromatin. CONCLUSIONS: Our results demonstrate that Anopheles polytene chromosomes and whole-genome shotgun assembly render the mapping and characterization of a significant part of heterochromatic scaffolds a possibility. These results reveal the strong association between characteristics of the genome features and morphological types of chromatin. Initial analysis of the An. gambiae heterochromatin provides a framework for its functional characterization and comparative genomic analyses with other organisms.

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Using paired-end sequences to optimise parameters for alignment of sequence reads against related genomes.

Publication Date: 2010 PMID: 20678236
Authors: Ratnakumar, A. - McWilliam, S. - Barris, W. - Dalrymple, B. P.
Journal: BMC Genomics

BACKGROUND: The advent of cheap high through-put sequencing methods has facilitated low coverage skims of a large number of organisms. To maximise the utility of the sequences, assembly into contigs and then ordering of those contigs is required. Whilst sequences can be assembled into contigs de novo, using assembled genomes of closely related organisms as a framework can considerably aid the process. However, the preferred search programs and parameters that will optimise the sensitivity and specificity of the alignments between the sequence reads and the framework genome(s) are not necessarily obvious. Here we demonstrate a process that uses paired-end sequence reads to choose an optimal program and alignment parameters. RESULTS: Unlike two single fragment reads, in paired-end sequence reads, such as BAC-end sequences, the two sequences in the pair have a known positional relationship in the original genome. This provides an additional level of confidence over match scores and e-values in the accuracy of the positional assignment of the reads in the comparative genome. Three commonly used sequence alignment programs: MegaBLAST, Blastz and PatternHunter were used to align a set of ovine BAC-end sequences against the equine genome assembly. A range of different search parameters, with a particular focus on contiguous and discontiguous seeds, were used for each program. The number of reads with a hit and the number of read pairs with hits for the two end sequences in the tail-to-tail paired-end configuration were plotted relative to the theoretical maximum expected curve. Of the programs tested, MegaBLAST with short contiguous seed lengths (word size 8-11) performed best in this particular task. In addition the data also provides estimates of the false positive and false negative rates, which can be used to determine the appropriate values of additional parameters, such as score cut-off, to balance sensitivity and specificity. To determine whether the approach also worked for the alignment of shorter reads, the first 240 bases of each BAC end sequence were also aligned to the equine genome. Again, contiguous MegaBLAST performed the best in optimising the sensitivity and specificity with which sheep BAC end reads map to the equine and bovine genomes. CONCLUSIONS: Paired-end reads, such as BAC-end sequences, provide an efficient mechanism to optimise sequence alignment parameters, for example for comparative genome assemblies, by providing an objective standard to evaluate performance.

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