| 1. |
- Alexeyenko, Andrey, et al.
(författare)
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Efficient de novo assembly of large and complex genomes by massively parallel sequencing of Fosmid pools
- 2014
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 15, s. 439-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Sampling genomes with Fosmid vectors and sequencing of pooled Fosmid libraries on the Illumina platform for massive parallel sequencing is a novel and promising approach to optimizing the trade-off between sequencing costs and assembly quality. Results: In order to sequence the genome of Norway spruce, which is of great size and complexity, we developed and applied a new technology based on the massive production, sequencing, and assembly of Fosmid pools (FP). The spruce chromosomes were sampled with similar to 40,000 bp Fosmid inserts to obtain around two-fold genome coverage, in parallel with traditional whole genome shotgun sequencing (WGS) of haploid and diploid genomes. Compared to the WGS results, the contiguity and quality of the FP assemblies were high, and they allowed us to fill WGS gaps resulting from repeats, low coverage, and allelic differences. The FP contig sets were further merged with WGS data using a novel software package GAM-NGS. Conclusions: By exploiting FP technology, the first published assembly of a conifer genome was sequenced entirely with massively parallel sequencing. Here we provide a comprehensive report on the different features of the approach and the optimization of the process. We have made public the input data (FASTQ format) for the set of pools used in this study: ftp://congenie.org/congenie/Nystedt_2013/Assembly/ProcessedData/FosmidPools/.(alternatively accessible via http://congenie.org/downloads).The software used for running the assembly process is available at http://research.scilifelab.se/andrej_alexeyenko/downloads/fpools/.
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| 2. |
- Båge, Tove, et al.
(författare)
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Signal pathways JNK and NF-kappa B, identified by global gene expression profiling, are involved in regulation of TNF alpha-induced mPGES-1 and COX-2 expression in gingival fibroblasts
- 2010
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 11, s. 241-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Prostaglandin E-2 (PGE(2)) is involved in several chronic inflammatory diseases including periodontitis, which causes loss of the gingival tissue and alveolar bone supporting the teeth. We have previously shown that tumor necrosis factor a (TNF alpha) induces PGE(2) synthesis in gingival fibroblasts. In this study we aimed to investigate the global gene expression profile of TNF alpha-stimulated primary human gingival fibroblasts, focusing on signal pathways related to the PGE(2)-synthesizing enzymes prostaglandin E synthases (PGES), as well as the upstream enzyme cyclooxygenase-2 (COX-2) and PGE(2) production. Results: Microarray and western blot analyses showed that the mRNA and protein expression of the inflammatory induced microsomal prostaglandin E synthase-1 (mPGES-1) was up-regulated by the cytokine TNF alpha, accompanied by enhanced expression of COX-2 and increased production of PGE(2). In contrast, the expression of the isoenzymes microsomal prostaglandin E synthase-2 (mPGES-2) and cytosolic prostaglandin E synthase (cPGES) was unaffected by TNF alpha treatment. Using oligonucleotide microarray analysis in a time-course factorial design including time points 1, 3 and 6 h, differentially expressed genes in response to TNF alpha treatment were identified. Enrichment analysis of microarray data indicated two positively regulated signal transduction pathways: c-Jun N-terminal kinase (JNK) and Nuclear Factor-kappa B (NF-kappa B). To evaluate their involvement in the regulation of mPGES-1 and COX-2 expression, we used specific inhibitors as well as phosphorylation analysis. Phosphorylation analysis of JNK (T183/Y185) and NF-kappa B p65 (S536) showed increased phosphorylation in response to TNF alpha treatment, which was decreased by specific inhibitors of JNK (SP600125) and NF-kappa B (Bay 11-7082, Ro 106-9920). Inhibitors of JNK and NF-kappa B also decreased the TNF alpha-stimulated up-regulation of mPGES-1 and COX-2 as well as PGE(2) production. Conclusion: In the global gene expression profile, the enrichment analysis of microarray data identified the two signal transduction pathways JNK and NF-kappa B as positively regulated by the cytokine TNF alpha. Inhibition of these TNF alpha-activated signal pathways reduced the expression of mPGES-1 and COX-2 as well as their end product PGE(2) in gingival fibroblasts. The involvement of the signal pathways JNK and NF-kappa B in the regulation of PGE(2) induced by TNF alpha may suggest these two pathways as possible attractive targets in the chronic inflammatory disease periodontitis.
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| 3. |
- Klevebring, Daniel, 1981-, et al.
(författare)
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Analysis of transcript and protein overlap in a human osteosarcoma cell line
- 2010
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 11:1, s. 684-
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Tidskriftsartikel (refereegranskat)abstract
- Background: An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq) to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC) and immunofluorescence microscopy (IF). Results: A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2%) were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies. Conclusion: This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.
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| 4. |
- Klevebring, Daniel, 1981-, et al.
(författare)
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Genome-wide profiling of Populus small RNAs
- 2009
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 10, s. Article number 620-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced Populus trichocarpa using a concatenation strategy in combination with 454 sequencing. Results: The most abundant size class of sRNAs were 24 nt and these were generally associated with a number of classes of retrotransposons and repetitive elements. Some repetitive elements were also associated with 22 nt RNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the sex-determining loci and a major cluster of NBS-LRR genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and NBS-LRR disease resistance genes, classes of genes that have been significantly expanded in Populus. Additional loci enriched for sRNA production were identified. We identified 15 novel predicted microRNAs (miRNAs), including miRNA∗ sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs). Conclusions: The short RNA population of P. trichocarpa is at least as complex as that of Arabidopsis. We provide a first genome-wide view of short RNA production for P. trichocarpa and identify new, non-conserved miRNAs.
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| 5. |
- Kutsenko, Alexey, et al.
(författare)
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The Chironomus tentans genome sequence and the organization of the Balbiani ring genes
- 2014
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 15, s. 819-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The polytene nuclei of the dipteran Chironomus tentans (Ch. tentans) with their Balbiani ring (BR) genes constitute an exceptional model system for studies of the expression of endogenous eukaryotic genes. Here, we report the first draft genome of Ch. tentans and characterize its gene expression machineries and genomic architecture of the BR genes. Results: The genome of Ch. tentans is approximately 200 Mb in size, and has a low GC content (31%) and a low repeat fraction (15%) compared to other Dipteran species. Phylogenetic inference revealed that Ch. tentans is a sister clade to mosquitoes, with a split 150-250 million years ago. To characterize the Ch. tentans gene expression machineries, we identified potential orthologus sequences to more than 600 Drosophila melanogaster (D. melanogaster) proteins involved in the expression of protein-coding genes. We report novel data on the organization of the BR gene loci, including a novel putative BR gene, and we present a model for the organization of chromatin bundles in the BR2 puff based on genic and intergenic in situ hybridizations. Conclusions: We show that the molecular machineries operating in gene expression are largely conserved between Ch. tentans and D. melanogaster, and we provide enhanced insight into the organization and expression of the BR genes. Our data strengthen the generality of the BR genes as a unique model system and provide essential background for in-depth studies of the biogenesis of messenger ribonucleoprotein complexes.
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| 6. |
- Käller, Max, et al.
(författare)
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Comparison of PrASE and Pyrosequencing for SNP Genotyping
- 2006
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 7, s. 291-
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers. Results: The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated. Conclusion: The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.
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| 7. |
- Richter, Karin, et al.
(författare)
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Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
- 2006
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 7, s. 75-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types.Results: Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development.Conclusion: Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.
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| 8. |
- Richter, Karin, et al.
(författare)
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Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
- 2006
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 7, s. 75-
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Tidskriftsartikel (refereegranskat)abstract
- Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.
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| 9. |
- Rubin, Carl-Johan, et al.
(författare)
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Differential gene expression in femoral bone from red junglefowl and domestic chicken, differing for bone phenotypic traits
- 2007
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 8, s. 208-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Osteoporosis is frequently observed among aging hens from egg-producing strains (layers) of domestic chicken. White Leghorn (WL) has been intensively selected for egg production and it manifests striking phenotypic differences for a number of traits including several bone phenotypes in comparison with the wild ancestor of chicken, the red junglefowl (RJ). Previously, we have identified four Quantitative Trait Loci (QTL) affecting bone mineral density and bone strength in an intercross between RJ and WL. With the aim of further elucidating the genetic basis of bone traits in chicken, we have now utilized cDNA-microarray technology in order to compare global RNA-expression in femoral bone from adult RJ and WL (five of each sex and population). Results: When contrasting microarray data for all WL-individuals to that of all RJ-individuals we observed differential expression (False discovery rate adjusted p-values < 0.015) for 604 microarray probes. In corresponding male and female contrasts, differential expression was observed for 410 and 270 probes, respectively. Altogether, the three contrasts between WL and RJ revealed differential expression of 779 unique transcripts, 57 of which are located to previously identified QTL-regions for bone traits. Some differentially expressed genes have previously been attributed roles in bone metabolism and these were: WNT inhibitory factor I (WIFI), WD repeat-containing protein 5 (WDR5) and Syndecan 3 (SDC3). Among differentially expressed transcripts, those encoding structural ribosomal proteins were highly enriched and all 15 had lower expression in WL. Conclusion: We report the identification of 779 differentially expressed transcripts, several residing within QTL-regions for bone traits. Among differentially expressed transcripts, those encoding structural ribosomal proteins were highly enriched and all had lower expression levels in WL. In addition, transcripts encoding four translation initiation and translation elongation factor proteins also had lower expression levels in WL, possibly indicating perturbation of protein biosynthesis pathways between the two populations. Information derived from this study could be relevant to the bone research field and may also aid in further inference of genetic changes accompanying animal domestication.
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| 10. |
- Sandberg, Julia, et al.
(författare)
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Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing
- 2010
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 11:140
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Tidskriftsartikel (refereegranskat)abstract
- Background In addition to shotgun sequencing, next generation sequencing has been shown to be suitable for deep sequencing of many specific PCR-amplified target genes in parallel. However, unspecific product formation is a common problem in amplicon sequencing since these fragments are difficult to fully remove by gel purification, and their presence inevitably reduces the number of mappable sequence reads that can be obtained in each sequencing run. Results We have used a novel flow cytometric sorting approach to specifically enrich Roche/454 DNA Capture beads carrying target DNA sequences on their surface, and reject beads carrying unspecific sequences. This procedure gives a nearly three-fold increase in the fraction of informative sequences obtained. Presented results also show that there are no significant differences in the distribution or presence of different genotypes between a FACS-enriched sample and a standard-enriched control sample. Conclusions Target-specific FACS enrichment prior to Roche/454 sequencing provides a quick, inexpensive way of increasing the amount of high quality data obtained in a single sequencing run, without introducing any sequence bias.
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