Expanding the catalog of cas genes with metagenomes.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhang Q
PROVIDER: S-EPMC3936711 | biostudies-literature | 2014 Feb
REPOSITORIES: biostudies-literature
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Project description:While the majority of protein-coding genes in the human genome have been identified, the location of the regulatory sequences that control their expression in time and space remains poorly defined. The importance of identifying these elements is underscored by a growing number of studies (such as GWAS) supporting a relationship between variation in non-coding sequences and human disease. M-BM- emonstrated that ChIP-Seq with p300 directly on human or mouse tissues represents a powerful method to identify the location and tissue-specific activity pattern of enhancers in the genome.M-BM- a highly specific chromatin mark for the prediction of in vivo enhancers, it is not without limitations. M-BM- comparison of p300-bound regions with previously generated enhancer sets shows that the majority of enhancers are activated independently of p300. To attempt to identify a larger proportion of enhancers in vivo, we have focused on several additional transcriptional coactivators that are hypothesized to be associated with active tissue-specific enhancers. M-BM- limited availability of ChIP-seq grade antibodies for these coactivators, FLAG-tag knock-in mice were generated and ChIP-seq using a FLAG antibody was performed on various tissues from e11.5 mouse embryos. Indeed, as these mouse lines have become available we have validated their valuable role as marks for transcriptional enhancers in vivo.M-BM- studies are expected to further expand the catalogue of in vivo functional enhancers, which will aid in the decoding of the regulatory genome and provide new insights into the role of gene regulation in human biology and disease. In addition to FLAG data, this accession includes histone acetylation / methylation ChIP-seq data from e11.5 mouse embryonic tissues. Histone ChIP-seq data was used in combination with FLAG data for various computational analyses. Examination of FLAG-labeled transcriptional coactivator binding in mouse embryonic stage 11.5 and embryonic stem cells
2014-04-16 | E-GEOD-37151 | biostudies-arrayexpress
Project description:While the majority of protein-coding genes in the human genome have been identified, the location of the regulatory sequences that control their expression in time and space remains poorly defined. The importance of identifying these elements is underscored by a growing number of studies (such as GWAS) supporting a relationship between variation in non-coding sequences and human disease. We previously demonstrated that ChIP-Seq with p300 directly on human or mouse tissues represents a powerful method to identify the location and tissue-specific activity pattern of enhancers in the genome. While p300 is a highly specific chromatin mark for the prediction of in vivo enhancers, it is not without limitations. In particular, comparison of p300-bound regions with previously generated enhancer sets shows that the majority of enhancers are activated independently of p300. To attempt to identify a larger proportion of enhancers in vivo, we have focused on several additional transcriptional coactivators that are hypothesized to be associated with active tissue-specific enhancers. To overcome the limited availability of ChIP-seq grade antibodies for these coactivators, FLAG-tag knock-in mice were generated and ChIP-seq using a FLAG antibody was performed on various tissues from e11.5 mouse embryos. Indeed, as these mouse lines have become available we have validated their valuable role as marks for transcriptional enhancers in vivo. These ongoing studies are expected to further expand the catalogue of in vivo functional enhancers, which will aid in the decoding of the regulatory genome and provide new insights into the role of gene regulation in human biology and disease. In addition to FLAG data, this accession includes histone acetylation / methylation ChIP-seq data from e11.5 mouse embryonic tissues. Histone ChIP-seq data was used in combination with FLAG data for various computational analyses.
2014-04-16 | GSE37151 | GEO
Project description:BackgroundMetagenome sampling bias for geographical location and lifestyle is partially responsible for the incomplete catalog of reference genomes of gut microbial species. Thus, genome assembly from currently under-represented populations may effectively expand the reference gut microbiome and improve taxonomic and functional profiling.MethodsWe assembled genomes using public whole-metagenomic shotgun sequencing (WMS) data for 110 and 645 fecal samples from India and Japan, respectively. In addition, we assembled genomes from newly generated WMS data for 90 fecal samples collected from Korea. Expecting genome assembly for low-abundance species may require a much deeper sequencing than that usually employed, so we performed ultra-deep WMS (> 30 Gbp or > 100 million read pairs) for the fecal samples from Korea. We consequently assembled 29,082 prokaryotic genomes from 845 fecal metagenomes for the three under-represented Asian countries and combined them with the Unified Human Gastrointestinal Genome (UHGG) to generate an expanded catalog, the Human Reference Gut Microbiome (HRGM).ResultsHRGM contains 232,098 non-redundant genomes for 5414 representative prokaryotic species including 780 that are novel, > 103 million unique proteins, and > 274 million single-nucleotide variants. This is an over 10% increase from the UHGG. The new 780 species were enriched for the Bacteroidaceae family, including species associated with high-fiber and seaweed-rich diets. Single-nucleotide variant density was positively associated with the speciation rate of gut commensals. We found that ultra-deep sequencing facilitated the assembly of genomes for low-abundance taxa, and deep sequencing (e.g., > 20 million read pairs) may be needed for the profiling of low-abundance taxa. Importantly, the HRGM significantly improved the taxonomic and functional classification of sequencing reads from fecal samples. Finally, analysis of human self-antigen homologs on the HRGM species genomes suggested that bacterial taxa with high cross-reactivity potential may contribute more to the pathogenesis of gut microbiome-associated diseases than those with low cross-reactivity potential by promoting inflammatory condition.ConclusionsBy including gut metagenomes from previously under-represented Asian countries, Korea, India, and Japan, we developed a substantially expanded microbiome catalog, HRGM. Information of the microbial genomes and coding genes is publicly available ( www.mbiomenet.org/HRGM/ ). HRGM will facilitate the identification and functional analysis of disease-associated gut microbiota.
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Project description:Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.
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Project description:BackgroundThe true diploid frog, Xenopus tropicalis (X. tropicalis) is an excellent genetic model organism. To date, the CRISPR/Cas-mediated genome editing methods established in this species are mostly based on SpCas9 that requires the stringent NGG protospacer-adjacent motif (PAM) for target recognition, which limits its genome editing scope. Thus, it is highly desirable to circumvent this limitation.ResultsThrough one-cell stage injection of Cas/gRNAs into X. tropicalis embryos, we evaluated the mutagenic efficiency of 8 different Cas variants using T7EI assay, Sanger DNA sequencing, or deep sequencing. Our data indicate that SaCas9 and KKH SaCas9 are highly effective in frogs, which could be used for direct phenotyping in G0 embryos. In contrast, VQR Cas9, xCas9 3.7, SpG Cas9, and SpRY Cas9 were ineffective in X. tropicalis embryos and no activity was detected for iSpyMac Cas9. We also found that LbCas12a/crRNA RNP complexes with paired crRNAs efficiently induced small fragment deletions in X. tropicalis embryos.ConclusionSaCas9 and KKH SaCas9 are robust genome editing tools in X. tropicalis embryos. LbCas12a/crRNA RNP complexes are useful for inducing DNA fragment deletions in frog embryos. These tools expand the CRISPR/Cas genome editing scope in X. tropicalis and increase the flexibility for various genome editing applications in frogs.
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