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De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.)
Background:
In rubber tree, the bark is an important agricultural and biological organ. However, the molecular mechanism involved in the bark formation and development in rubber tree remains largely unknown, which is at least partially due to lack of bark transcriptomic and genomic information. Therefore, high-throughput transcriptome sequencing of rubber tree bark is carried out to generate enormous transcript sequences for the transcriptome characterization and molecular marker development.
Results:
In this study, more than 30 million sequencing reads were generated using Illumina paired-end sequencing technology. In total, 22,756 unigenes with an average length of 485bp were obtained by de novo assembly. The similarity search indicated that 16,520 and 12,558 unigenes showed significant similarities to known proteins from NCBI non-redundant and Swissprot protein databases, respectively. Among these annotated unigenes, 6,867 and 5,559 unigenes were separately assigned to Gene Ontology (GO) and Clusters of Orthologous Group (COG). By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 12,097 unigenes were assigned to 5 main categories including 123 KEGG pathways. Among the main KEGG categories, metabolism was the biggest category (9,043, 74.75%), indicating that the active metabolic processes in rubber tree bark. In addition, a total of 39,257 EST-SSRs were predicted as within 22,756 unigenes, and the characterizations of EST-SSRs were further analyzed in rubber tree. 110 potential marker sites were randomly selected to validate the assembly quality and develop new markers. The result showed that PCR success rate and polymorphism rate of 110 markers were separately 96.36% and 55.45% among 13 Hevea germplasms.
Conclusion:
By assembling and analyzing de novo transcriptome sequencing data, we reported the transcriptomic characterization of rubber tree bark in detail. This research generated a substantial fraction of rubber tree transcriptome sequences, which were very useful resources for gene annotation and discovery, molecular markers development, genome assembly and annotation, and microarrays development in H. brasiliensis. The EST-SSR markers predicted in this study might enrich molecular markers and facilitate marker-assisted selection breeding in rubber tree. Moreover, this study also supported that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for transcriptome characterization and molecular marker development in non-model species, especially those with large and complex genome.
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Comparative genome analysis of central nitrogen metabolism and its control by GlnR in the class Bacilli
Background:
The assimilation of nitrogen in bacteria is achieved through only a few central chemical reactions. In low-GC Gram-positive bacteria the transcriptional control over the levels of the related enzymes is mediated by four regulators: GlnR, TnrA, GltC and CodY. We have analyzed the genomes of all species belonging to the taxonomic families Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae to determine the diversity in central nitrogen metabolism and reconstructed the regulation by GlnR.
Results:
Although we observed a substantial difference in the extent of central nitrogen metabolism in the various species, the basic GlnR regulon was remarkably constant and appeared not affected by the presence or absence of the other two main regulators. We found a conserved regulatory association of GlnR with glutamine synthetase (glnRA operon), and the active transport of ammonium (amtB-glnK) and glutamine/glutamate (i.e. via glnQHMP, glnPHQ, gltT, alsT). In addition less-conserved associations were found with, for instance, glutamate dehydrogenase in Streptococcaceae, purine catabolism and the reduction of nitrite in Bacillaceae, and aspartate/asparagine deamination in Lactobacillaceae.
Conclusions:
Our analyses imply GlnR-mediated regulation in constraining the import of ammonia/amino-containing compounds and the production of intracellular ammonia under conditions of high nitrogen availability. Such a role fits with the intrinsic need for tight control of ammonia levels to limit futile cycling.
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Comparison of low and high dose ionising radiation using topological analysis of gene coexpression networks
The growing use of imaging procedures in medicine has raisedconcerns about exposure to low-dose ionising radiation (LDIR). Whilethe disastrous effects of high dose ionising radiation (HDIR) iswell documented, the detrimental effects of LDIR is not wellunderstood and has been a topic of much debate. Since little isknown about the effects of LDIR, various kinds of wet-lab andcomputational analyses are required to advance knowledge in thisdomain. In this paper we carry out an "upside-down pyramid" form ofsystems biology analysis of microarray data. We characterised theglobal genomic response following 10 cGy (low dose) and 100 cGy(high dose) doses of X-ray ionising radiation at four time points byanalysing the topology of gene coexpression networks. This studyincludes a rich experimental design and state-of-the-artcomputational systems biology methods of analysis to study thedifferences in the transcriptional response of skin cells exposed tolow and high doses of radiation. Using this method we foundimportant genes that have been linked to immune response, cellsurvival and apoptosis. Furthermore, we also were able to identifygenes such as BRCA1, ABCA1, TNFRSF1B, MLLT11 thats have beenassociated with various types of cancers. Our method of applyingnetwork topological differences can aid in identifying thedifferences among similar (eg: radiation effect) yet very differentbiological conditions (eg: different dose and time) to generatetestable hypotheses. This is the first study where a network levelanalysis was performed across two different radiation doses atvarious time points, thereby illustrating changes in the cellularresponse over time.
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Genome-wide recruitment to Polycomb-modified chromatin and activity regulation of the synovial sarcoma oncogene SYT-SSX2
Background:
SYT-SSX is the oncogene associated with synovial sarcoma (SS), a stem cell disease. SYT-SSX is thought to be responsible for sarcoma initiation and development. It interacts with components of Polycomb and SWI/SNF complexes, the two epigenetic controllers that maintain the heritable status of differentiation-specific genes in the stem/progenitor cell. Through these associations SYT-SSX is thought to alter gene expression programs by epigenetic mechanisms. Recently, we reported that SYT-SSX2 reprograms mesenchymal stem cells and myoblasts by dictating their commitment to the neural lineage while disrupting their normal differentiation. This reprogramming was due to the direct occupancy of proneural genes by the SYT-SSX2 nuclear complex. To gain a clear understanding of SYT-SSX2 control of gene expression networks, we conducted a thorough genome-wide analysis to determine the mechanism of its recruitment and identify signature sets of epigenetic markers that would predict its targeting and transcriptional activity.
Results:
SYT-SSX2 was recruited to distinct loci across all chromosomes, and an overwhelming number of Polycomb-modified sites enriched with the trimethylated histone H3 on lysine 27 (H3K27me3) formed the main recruiting module for SYT-SSX2. Not all SYT-SSX2/ H3K27me3-occupied genes had altered expression, denoting the requirement for additional signals upon oncogene binding. Differential binding and epigenetic patterns distinguished upregulated and downregulated genes. Most activated genes had SYT-SSX2 sites enriched with H3K27me3 within their body or near their transcription start site (TSS) whereas a majority of downregulated genes were characterized by SYT-SSX2/H3K27me3-rich regions at long-range, or by modifications associated with transcription activation within the gene body or near the TSS. Hierarchical and functional clustering identified H3K27me3 as the dominant epigenetic marker associated with SYT-SSX2 binding and gene expression. Notably, this analysis revealed a cluster of upregulated neuronal genes densely covered by H3K27me3, consistent with programming toward the neural lineage by SYT-SSX2 observed previously.
Conclusions:
the data analysis revealed that Polycomb complexes or their modified chromatin and their stably silenced differentiation programs seem to be the main target for SYT-SSX2, suggesting that their perturbation is at the center of tumorigenesis driven by the oncogene. Further research into this mechanism is crucial to the full understanding of SS biology.
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Differences in DNA curvature-related sequence periodicity between prokaryotic chromosomes and phages, and relationship to chromosomal prophage content
Background:
Periodic spacing of A-tracts (short runs of A or T) with the DNA helical period of ~10-11 bp is characteristic of intrinsically bent DNA. In eukaryotes, the DNA bending is related to chromatin structure and nucleosome positioning. However, the physiological role of strong sequence periodicity detected in many prokaryotic genomes is not clear.
Results:
We developed measures of intensity and persistency of DNA curvature-related sequence periodicity and applied them to prokaryotic chromosomes and phages. The results indicate that strong periodic signals present in chromosomes are generally absent in phage genomes. Moreover, chromosomes containing prophages are less likely to possess a persistent periodic signal than chromosomes with no prophages.
Conclusions:
Absence of DNA curvature-related sequence periodicity in phages could arise from constraints associated with DNA packaging in the viral capsid. Lack of prophages in chromosomes with persistent periodic signal suggests that the sequence periodicity and concomitant DNA curvature could play a role in protecting the chromosomes from integration of phage DNA.
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Genetic and genome-wide transcriptomic analyses
identify co-regulation of oxidative response and
hormone transcript abundance with vitamin c
content in tomato fruit
Background:
L-ascorbic acid (AsA; vitamin C) is essential for all living plants where it functions as themain hydrosoluble antioxidant. It has diverse roles in the regulation of plant cell growth andexpansion, photosynthesis, and hormone-regulated processes. AsA is also an essentialcomponent of the human diet, being tomato fruit one of the main sources of this vitamin. Toidentify genes responsible for AsA content in tomato fruit, transcriptomic studies followed byclustering analysis were applied to two groups of fruits with contrasting AsA content. Thesefruits were identified after AsA profiling of an F8 Recombinant Inbred Line (RIL) populationgenerated from a cross between the domesticated species Solanum lycopersicum and the wildrelative Solanum pimpinellifollium.
Results:
We found large variability in AsA content within the RIL population with individual RILswith up to 4-fold difference in AsA content. Transcriptomic analysis identified genes whoseexpression correlated either positively (PVC genes) or negatively (NVC genes) with the AsAcontent of the fruits. Cluster analysis using SOTA allowed the identification of subsets of coregulatedgenes mainly involved in hormones signaling, such as ethylene, ABA, gibberellinand auxin, rather than any of the known AsA biosynthetic genes. Data mining of thecorresponding PVC and NVC orthologs in Arabidopis databases identified flagellin and otherROS-producing processes as cues resulting in differential regulation of a high percentage ofthe genes from both groups of co-regulated genes; more specifically, 26.6% of theorthologous PVC genes, and 15.5% of the orthologous NVC genes were induced andrepressed, respectively, under flagellin22 treatment in Arabidopsis thaliana.
Conclusion:
Results here reported indicate that the content of AsA in red tomato fruit from our selectedRILs are not correlated with the expression of genes involved in its biosynthesis. On the contrary, the data presented here supports that AsA content in tomato fruit co-regulates withgenes involved in hormone signaling and they are dependent on the oxidative status of thefruit.
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Estimating RNA-quality using GeneChip microarrays
Background:
Microarrays are a powerful tool for transcriptome analysis. Best results are obtained using high-quality RNA samples for preparation and hybridization. Issues with RNA integrity can lead to low data quality and failure of the microarray experiment.
Results:
Microarray intensity data contains information to estimate the RNA quality of the sample. We here study the interplay of the characteristics of RNA surface hybridization with the effects of partly truncated transcripts on probe intensity. The 3'/5' intensity gradient, the basis of microarray RNA quality measures, is shown to depend on the degree of competitive binding of specific and of non-specific targets to a particular probe, on the degree of saturation of the probes with bound transcripts and on the distance of the probe from the 3'-end of the transcript. Increasing degrees of non-specific hybridization or of saturation reduce the 3'/5' intensity gradient and if not taken into account, this leads to biased results in common quality measures for GeneChip arrays such as affyslope or the control probe intensity ratio. We also found that short probe sets near the 3'-end of the transcripts are prone to non-specific hybridization presumable because of inaccurate positional assignment and the existence of transcript isoforms with variable 3' UTR's . Poor RNA quality is associated with a decreased amount of RNA material hybridized on the array paralleled by a decreased total signal level. Additionally, it causes a gene-specific loss of signal due to the positional bias of transcript abundance which requires an individual, gene-specific correction. We propose a new RNA quality measure that considers the hybridization mode. Graphical characteristics are introduced allowing assessment of RNA quality of each single array ('tongs plot' and 'degradation hook'). Furthermore, we suggest a method to correct for effects of RNA degradation on microarray intensities.
Conclusions:
The presented RNA degradation measure has best correlation with the independent RNA integrity measure RIN, and therefore presents itself as a valuable tool for quality control and even for the study of RNA degradation. When RNA degradation effects are detected in microarray experiments, a correction of the induced bias in probe intensities is advised.
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Development and application of a 6.5 million feature
affymetrix genechip® for massively parallel discovery of
single position polymorphisms in lettuce (Lactuca spp.)
Background:
High-resolution genetic maps are needed in many crops to help characterize the geneticdiversity that determines agriculturally important traits. Hybridization to microarrays todetect single feature polymorphisms is a powerful technique for marker discovery andgenotyping because of its highly parallel nature. However, microarrays designed for geneexpression analysis rarely provide sufficient gene coverage for optimal detection ofnucleotide polymorphisms, which limits utility in species with low rates of polymorphismsuch as lettuce (Lactuca sativa).
Results:
We developed a 6.5 million feature Affymetrix GeneChip® for efficient polymorphismdiscovery and genotyping, as well as for analysis of gene expression in lettuce. Probes on themicroarray were designed from 26,809 unigenes from cultivated lettuce and an additional8,819 unigenes from four related species (L. serriola, L. saligna, L. virosa and L. perennis).Where possible, probes were tiled with a 2 bp stagger, alternating on each DNA strand;providing an average of 187 probes covering approximately 600 bp for each of over 35,000unigenes; resulting in up to 13 fold redundancy in coverage per nucleotide. We developedprotocols for hybridization of genomic DNA to the GeneChip® and refined customalgorithms that utilized coverage from multiple, high quality probes to detect single positionpolymorphisms in 2 bp sliding windows across each unigene. This allowed us to detectgreater than 18,000 polymorphisms between the parental lines of our core mappingpopulation, as well as numerous polymorphisms between cultivated lettuce and wild speciesin the lettuce genepool. Using marker data from our diversity panel comprised of 52accessions from the five species listed above, we were able to separate accessions by speciesusing both phylogenetic and principal component analyses. Additionally, we estimated thediversity between different types of cultivated lettuce and distinguished morphological types.
Conclusion:
By hybridizing genomic DNA to a custom oligonucleotide array designed for maximum genecoverage, we were able to identify polymorphisms using two approaches for pair-wise
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Transcription profiling reveals stage- and functiondependent expression patterns in the filarial
nematode brugia malayi
Background:
Brugia malayi is a nematode parasite that causes lymphatic filariasis, a disfiguring anddisabiling tropical disease. Although a first draft genome sequence was released in 2007, verylittle is understood about transcription programs that govern developmental changes requiredfor the parasite's development and survival in its mammalian and insect hosts.
Results:
We used a microarray with probes that represent some 85% of predicted genes to generategene expression profiles for seven parasite life cycle stages/sexes. Approximately 41% oftranscripts with detectable expression signals were differentially expressed across lifecyclestages. Twenty-six percent of transcripts were exclusively expressed in a single parasite stage, and 27% were expressed in all stages studied. K-means clustering of differentiallyexpressed transcripts revealed five major transcription patterns that were associated withparasite lifecycle stages or gender. Examination of known stage-associated transcriptsvalidated these data sets and suggested that newly identified stage or gender-associatedtranscripts may exercise biological functions in development and reproduction. The resultsalso indicate that genes with similar transcription patterns were often involved in similarfunctions or cellular processes. For example, nuclear receptor family gene transcripts wereupregulated in gene expression pattern four (female-enriched) while protein kinase genefamily transcripts were upregulated in expression pattern five (male-enriched). We also usedpair-wise comparisons to identify transcriptional changes between life cycle stages and sexes.
Conclusions:
Analysis of gene expression patterns of lifecycle in B. malayi has provided novel insights intothe biology of filarial parasites. Proteins encoded by stage-associated and/or stage-specifictranscripts are likely to be critically important for key parasite functions such asestablishment and maintenance of infection, development, reproduction, and survival in thehost. Some of these may be useful targets for vaccines or new drug treatments for filariasis.
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Identification of avian W-linked contigs by short-read sequencing
Background:
The female-specific W chromosomes and male-specific Y chromosomes have proven difficult to assemble with whole-genome shotgun methods, creating a demand for new approaches to identify sequence contigs specific to these sex chromosomes. Here, we develop and apply a novel method for identifying sequences that are W-specific.
Results:
Using the Illumina Genome Analyzer, we generated sequence reads from a male domestic chicken (ZZ) and mapped them to the existing female (ZW) genome sequence. This method allowed us to identify segments of the female genome that are underrepresented in the male genome and are therefore likely to be female specific. We developed a Bayesian classifier to automate the calling of W-linked contigs and successfully identified more than 60 novel W-specific sequences.
Conclusions:
Our classifier can be applied to improve heterogametic whole-genome shotgun assemblies of the W or Y chromosome of any organism. This study greatly improves our knowledge of the W chromosome and will enhance future studies of avian sex determination and sex chromosome evolution.
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