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Sample records for genome based cell

  1. Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics.

    PubMed

    Rinke, Christian; Lee, Janey; Nath, Nandita; Goudeau, Danielle; Thompson, Brian; Poulton, Nicole; Dmitrieff, Elizabeth; Malmstrom, Rex; Stepanauskas, Ramunas; Woyke, Tanja

    2014-05-01

    Single-cell genomics is a powerful tool for exploring the genetic makeup of environmental microorganisms, the vast majority of which are difficult, if not impossible, to cultivate with current approaches. Here we present a comprehensive protocol for obtaining genomes from uncultivated environmental microbes via high-throughput single-cell isolation by FACS. The protocol encompasses the preservation and pretreatment of differing environmental samples, followed by the physical separation, lysis, whole-genome amplification and 16S rRNA-based identification of individual bacterial and archaeal cells. The described procedure can be performed with standard molecular biology equipment and a FACS machine. It takes <12 h of bench time over a 4-d time period, and it generates up to 1 μg of genomic DNA from an individual microbial cell, which is suitable for downstream applications such as PCR amplification and shotgun sequencing. The completeness of the recovered genomes varies, with an average of ∼50%.

  2. Baculovirus-based genome editing in primary cells.

    PubMed

    Mansouri, Maysam; Ehsaei, Zahra; Taylor, Verdon; Berger, Philipp

    2017-01-22

    Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template. Recombinant Lentivirus or Adenovirus genomes have enough capacity for a nuclease coding sequence and the gRNA but are usually too small to also carry large targeting constructs. We recently showed that a baculovirus-based multigene expression system (MultiPrime) can be used for genome editing in primary cells since it possesses the necessary capacity to carry the nuclease and gRNA expression constructs and the HDR targeting sequences. Here we present new Acceptor plasmids for MultiPrime that allow simplified cloning of baculoviruses for genome editing and we show their functionality in primary cells with limited life span and induced pluripotent stem cells (iPS).

  3. Functional cell-based uHTS in chemical genomic drug discovery.

    PubMed

    Croston, Glenn E

    2002-03-01

    The availability of genomic information significantly increases the number of potential targets available for drug discovery, although the function of many targets and their relationship to disease is unknown. In a chemical genomic research approach, ultra-high throughput screening (uHTS) of genomic targets takes place early in the drug discovery process, before target validation. Target-selective modulators then provide drug leads and pharmacological research tools to validate target function. Effective implementation of a chemical genomic strategy requires assays that can perform uHTS for large numbers of genomic targets. Cell-based functional assays are capable of the uHTS throughput required for chemical genomic research, and their functional nature provides distinct advantages over ligand-binding assays in the identification of target-selective modulators.

  4. Single-cell genomics-based analysis of virus–host interactions in marine surface bacterioplankton

    DOE PAGES

    Labonté, Jessica M.; Swan, Brandon K.; Poulos, Bonnie; ...

    2015-04-07

    Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. Furthermore, a combination of comparative genomics, metagenomic fragmentmore » recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. This study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities.« less

  5. Single-cell genomics-based analysis of virus–host interactions in marine surface bacterioplankton

    SciTech Connect

    Labonté, Jessica M.; Swan, Brandon K.; Poulos, Bonnie; Luo, Haiwei; Koren, Sergey; Hallam, Steven J.; Sullivan, Matthew B.; Woyke, Tanja; Eric Wommack, K.; Stepanauskas, Ramunas

    2015-04-07

    Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. Furthermore, a combination of comparative genomics, metagenomic fragment recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. This study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities.

  6. Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells.

    PubMed

    Kime, Cody; Mandegar, Mohammad A; Srivastava, Deepak; Yamanaka, Shinya; Conklin, Bruce R; Rand, Tim A

    2016-01-01

    Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)-cell-like properties has led to hPS cells with disease-specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments utilizing site-specific double-strand break (DSB)-mediated genome engineering tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPS cells. However, these methods are technically cumbersome and require significant expertise, which has limited adoption. A major recent advance involving the clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPS cells. In this unit, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPS cells into cell lines containing genomes altered by insertion/deletion (indel) mutagenesis or insertion of recombinant genomic DNA.

  7. Genome engineering in human cells.

    PubMed

    Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

  8. Efficient CRISPR/Cas9-based Genome Engineering in Human Pluripotent Stem Cells

    PubMed Central

    Kime, Cody; Mandegar, Mohammad A.; Srivastava, Deepak; Yamanaka, Shinya; Conklin, Bruce R.; Rand, Tim A.

    2016-01-01

    Human pluripotent stem cells (hPSCs) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem (hiPS) cells with human embryonic stem (hES) cell-like properties has led to hPSCs with disease-specific genetic backgrounds for in-vitro disease modeling, drug discovery, mechanistic and developmental studies. To fully realize this potential it will be necessary to modify the genome of hPSCs with precision and flexibility. Pioneering experiments utilizing site-specific double strand break (DSB)-mediated genome engineering tools, including Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPSCs. However, these methods are technically cumbersome and require significant expertise, which limited adoption. A major recent advance involving the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPSCs. Herein, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPSCs to cell lines containing genomes altered by Insertion/Deletion (INDEL) mutagenesis or insertion of recombinant genomic DNA. PMID:26724721

  9. Comparative Analysis of Human B Cell Epitopes Based on BCG Genomes

    PubMed Central

    Liu, Haican; Zhao, Xiuqin; Wan, Kanglin

    2016-01-01

    Background. Tuberculosis is a huge global health problem. BCG is the only vaccine used for about 100 years against TB, but the reasons for protection variability in populations remain unclear. To improve BCG efficacy and develop a strategy for new vaccines, the underlying genetic differences among BCG subtypes should be understood urgently. Methods and Findings. Human B cell epitope data were collected from the Immune Epitope Database. Epitope sequences were mapped with those of 15 genomes, including 13 BCGs, M. bovis AF2122/97, and M. tuberculosis H37Rv, to identify epitopes distribution. Among 398 experimentally verified B cell epitopes, 321 (80.7%) were conserved, while the remaining 77 (19.3%) were lost to varying degrees in BCGs. The variable protective efficacy of BCGs may result from the degree of B cell epitopes deficiency. Conclusions. Here we firstly analyzed the genetic characteristics of BCGs based on B cell epitopes and found that B cell epitopes distribution may contribute to vaccine efficacy. Restoration of important antigens or effective B cell epitopes in BCG could be a useful strategy for vaccine development. PMID:27382565

  10. Chromosomal Instability Estimation Based on Next Generation Sequencing and Single Cell Genome Wide Copy Number Variation Analysis

    PubMed Central

    Dago, Angel E.; Leitz, Laura J.; Wang, Yipeng; Lee, Jerry; Werner, Shannon L.; Gendreau, Steven; Patel, Premal; Jia, Shidong; Zhang, Liangxuan; Tucker, Eric K.; Malchiodi, Michael; Graf, Ryon P.; Dittamore, Ryan; Marrinucci, Dena; Landers, Mark

    2016-01-01

    Genomic instability is a hallmark of cancer often associated with poor patient outcome and resistance to targeted therapy. Assessment of genomic instability in bulk tumor or biopsy can be complicated due to sample availability, surrounding tissue contamination, or tumor heterogeneity. The Epic Sciences circulating tumor cell (CTC) platform utilizes a non-enrichment based approach for the detection and characterization of rare tumor cells in clinical blood samples. Genomic profiling of individual CTCs could provide a portrait of cancer heterogeneity, identify clonal and sub-clonal drivers, and monitor disease progression. To that end, we developed a single cell Copy Number Variation (CNV) Assay to evaluate genomic instability and CNVs in patient CTCs. For proof of concept, prostate cancer cell lines, LNCaP, PC3 and VCaP, were spiked into healthy donor blood to create mock patient-like samples for downstream single cell genomic analysis. In addition, samples from seven metastatic castration resistant prostate cancer (mCRPC) patients were included to evaluate clinical feasibility. CTCs were enumerated and characterized using the Epic Sciences CTC Platform. Identified single CTCs were recovered, whole genome amplified, and sequenced using an Illumina NextSeq 500. CTCs were then analyzed for genome-wide copy number variations, followed by genomic instability analyses. Large-scale state transitions (LSTs) were measured as surrogates of genomic instability. Genomic instability scores were determined reproducibly for LNCaP, PC3, and VCaP, and were higher than white blood cell (WBC) controls from healthy donors. A wide range of LST scores were observed within and among the seven mCRPC patient samples. On the gene level, loss of the PTEN tumor suppressor was observed in PC3 and 5/7 (71%) patients. Amplification of the androgen receptor (AR) gene was observed in VCaP cells and 5/7 (71%) mCRPC patients. Using an in silico down-sampling approach, we determined that DNA copy

  11. Geosmin induces genomic instability in the mammalian cell microplate-based comet assay.

    PubMed

    Silva, Aline Flor; Lehmann, Mauricio; Dihl, Rafael Rodrigues

    2015-11-01

    Geosmin (GEO) (trans-1,10-dimethyl-trans-9-decalol) is a metabolite that renders earthy and musty taste and odor to water. Data of GEO genotoxicity on mammalian cells are scarce in the literature. Thus, the present study assessed the genotoxicity of GEO on Chinese hamster ovary (CHO) cells in the microplate-based comet assay. The percent of tail DNA (tail intensity (TI)), tail moment (TM), and tail length (TL) were used as parameters for DNA damage assessment. The results demonstrated that concentrations of GEO of 30 and 60 μg/mL were genotoxic to CHO cells after 4- and 24-h exposure periods, in all parameters evaluated, such as TI, TM, and TL. Additionally, GEO 15 μg/mL was genotoxic in the three parameters only in the 24-h exposure time. The same was observed for GEO 7.5 μg/mL, which induced significant DNA damage observed as TI in the 24-h treatment. The results present evidence that exposure to GEO may be associated with genomic instability in mammalian cells.

  12. One Bacterial Cell, One Complete Genome

    SciTech Connect

    Woyke, Tanja; Tighe, Damon; Mavrommatis, Konstantinos; Clum, Alicia; Copeland, Alex; Schackwitz, Wendy; Lapidus, Alla; Wu, Dongying; McCutcheon, John P.; McDonald, Bradon R.; Moran, Nancy A.; Bristow, James; Cheng, Jan-Fang

    2010-04-26

    While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200?900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  13. Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification

    PubMed Central

    Fu, Yusi; Li, Chunmei; Lu, Sijia; Zhou, Wenxiong; Tang, Fuchou; Xie, X. Sunney; Huang, Yanyi

    2015-01-01

    Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (105) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy. PMID:26340991

  14. Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification.

    PubMed

    Fu, Yusi; Li, Chunmei; Lu, Sijia; Zhou, Wenxiong; Tang, Fuchou; Xie, X Sunney; Huang, Yanyi

    2015-09-22

    Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (10(5)) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.

  15. Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool

    PubMed Central

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Hansen, Henning Gram; Kallehauge, Thomas Beuchert; Betenbaugh, Michael J; Nielsen, Alex Toftgaard; Kildegaard, Helene Faustrup

    2014-01-01

    Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells. Biotechnol. Bioeng. 2014; 111: 1604–1616. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:24827782

  16. Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool.

    PubMed

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Hansen, Henning Gram; Kallehauge, Thomas Beuchert; Betenbaugh, Michael J; Nielsen, Alex Toftgaard; Kildegaard, Helene Faustrup

    2014-08-01

    Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named "CRISPy" for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells.

  17. Cancer Cell Line Panels Empower Genomics-Based Discovery of Precision Cancer Medicine.

    PubMed

    Kim, Hyun Seok; Sung, Yeo-Jin; Paik, Soonmyung

    2015-09-01

    Since the first human cancer cell line, HeLa, was established in the early 1950s, there has been a steady increase in the number and tumor type of available cancer cell line models. Cancer cell lines have made significant contributions to the development of various chemotherapeutic agents. Recent advances in multi-omics technologies have facilitated detailed characterizations of the genomic, transcriptomic, proteomic, and epigenomic profiles of these cancer cell lines. An increasing number of studies employ the power of a cancer cell line panel to provide predictive biomarkers for targeted and cytotoxic agents, including those that are already used in clinical practice. Different types of statistical and machine learning algorithms have been developed to analyze the large-scale data sets that have been produced. However, much work remains to address the discrepancies in drug assay results from different platforms and the frequent failures to translate discoveries from cell line models to the clinic. Nevertheless, continuous expansion of cancer cell line panels should provide unprecedented opportunities to identify new candidate targeted therapies, particularly for the so-called "dark matter" group of cancers, for which pharmacologically tractable driver mutations have not been identified.

  18. Cancer Cell Line Panels Empower Genomics-Based Discovery of Precision Cancer Medicine

    PubMed Central

    Kim, Hyun Seok; Sung, Yeo-Jin

    2015-01-01

    Since the first human cancer cell line, HeLa, was established in the early 1950s, there has been a steady increase in the number and tumor type of available cancer cell line models. Cancer cell lines have made significant contributions to the development of various chemotherapeutic agents. Recent advances in multi-omics technologies have facilitated detailed characterizations of the genomic, transcriptomic, proteomic, and epigenomic profiles of these cancer cell lines. An increasing number of studies employ the power of a cancer cell line panel to provide predictive biomarkers for targeted and cytotoxic agents, including those that are already used in clinical practice. Different types of statistical and machine learning algorithms have been developed to analyze the large-scale data sets that have been produced. However, much work remains to address the discrepancies in drug assay results from different platforms and the frequent failures to translate discoveries from cell line models to the clinic. Nevertheless, continuous expansion of cancer cell line panels should provide unprecedented opportunities to identify new candidate targeted therapies, particularly for the so-called "dark matter" group of cancers, for which pharmacologically tractable driver mutations have not been identified. PMID:26256959

  19. Ultrahigh-throughput approach for analyzing single-cell genomic damage with an agarose-based microfluidic comet array.

    PubMed

    Li, Yiwei; Feng, Xiaojun; Du, Wei; Li, Ying; Liu, Bi-Feng

    2013-04-16

    Genomic DNA damage was generally identified with a "comet assay" but limited by low throughput and poor reproducibility. Here we demonstrated an ultrahigh-throughput approach with a microfluidic chip to simultaneously interrogate DNA damage conditions of up to 10,000 individual cells (approximately 100-fold in throughput over the conventional method) with better reproducibility. For experiment, agarose was chosen as the chip fabrication material, which would further act as an electrophoretic sieving matrix for DNA fragments separation. Cancer cells (HeLa or HepG2) were lined up in parallel microchannels by capillary effect to form a dense array of single cells. After treatment with different doses of hydrogen peroxide, individual cells were then lysed for subsequent single-cell gel electrophoresis in the direction vertical to microchannel and fluorescence detection. Through morphological analysis and fluorescent measurement of comet-shaped DNA, the damage conditions of individual cells could be quantified. DNA repair capacity was further evaluated to validate the reliability of this method. It indicated that the agarose-based microfluidic comet array electrophoresis was simple, highly reproducible, and of high throughput, providing a new method for highly efficient single-cell genomic analysis.

  20. Virus integration and genome influence in approaches to stem cell based therapy for andro-urology.

    PubMed

    Li, Longkun; Zhang, Deying; Li, Peng; Damaser, Margot; Zhang, Yuanyuan

    2015-03-01

    Despite the potential of stem cells in cell-based therapy, major limitations such as cell retention, ingrowth, and trans-differentiation after implantation remain. One technique for genetic modification of cells for tissue repair is the introduction of specific genes using molecular biology techniques, such as virus integration, to provide a gene that adds new functions to enhance cellular function, and to secrete trophic factors for recruiting resident cells to participate in tissue repair. Stem cells can be labeled to track cell survival, migration, and lineage. Increasing evidence demonstrates that cell therapy and gene therapy in combination remarkably improve differentiation of implanted mesenchymal stromal cells (MSCs), revascularization, and innervation in genitourinary tissues, especially to treat urinary incontinence, erectile dysfunction, lower urinary tract reconstruction, and renal failure. This review discusses the benefits, safety, side effects, and alternatives for using genetically modified MSCs in tissue regeneration in andro-urology.

  1. Virus Integration and Genome Influence in Approaches to Stem Cell Based Therapy for Andro-Urology

    PubMed Central

    Li, Longkun; Zhang, Deying; Li, Peng; Damaser, Margot; Zhang, Yuanyuan

    2014-01-01

    Despite the potential of stem cells in cell-based therapy, major limitations such as cell retention, ingrowth, and trans-differentiation after implantation remain. One technique for genetic modification of cells for tissue repair is the introduction of specific genes using molecular biology techniques, such as virus integration, to provide a gene that adds new functions to enhance cellular function, and to secrete trophic factors for recruiting resident cells to participate in tissue repair. Stem cells can be labelled to track cell survival, migration, and lineage. Increasing evidence demonstrates that cell therapy and gene therapy in combination remarkably improve myogenic differentiation of implanted mesenchymal stromal cells (MSCs), revascularization, and innervation in genitourinary tissues, especially to treat urinary incontinence, erectile dysfunction, lower urinary tract reconstruction, and renal failure. This review discusses the benefits, safety, side effects, and alternatives for using genetically modified MSCs in tissue regeneration in andro-urology. PMID:25453258

  2. Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

    PubMed

    Clark, Stephen J; Smallwood, Sébastien A; Lee, Heather J; Krueger, Felix; Reik, Wolf; Kelsey, Gavin

    2017-03-01

    DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

  3. Genomic expression differences between cutaneous cells from red hair color individuals and black hair color individuals based on bioinformatic analysis.

    PubMed

    Puig-Butille, Joan Anton; Gimenez-Xavier, Pol; Visconti, Alessia; Nsengimana, Jérémie; Garcia-García, Francisco; Tell-Marti, Gemma; Escamez, Maria José; Newton-Bishop, Julia; Bataille, Veronique; Del Río, Marcela; Dopazo, Joaquín; Falchi, Mario; Puig, Susana

    2016-12-24

    The MC1R gene plays a crucial role in pigmentation synthesis. Loss-of-function MC1R variants, which impair protein function, are associated with red hair color (RHC) phenotype and increased skin cancer risk. Cultured cutaneous cells bearing loss-of-function MC1R variants show a distinct gene expression profile compared to wild-type MC1R cultured cutaneous cells. We analysed the gene signature associated with RHC co-cultured melanocytes and keratinocytes by Protein-Protein interaction (PPI) network analysis to identify genes related with non-functional MC1R variants. From two detected networks, we selected 23 nodes as hub genes based on topological parameters. Differential expression of hub genes was then evaluated in healthy skin biopsies from RHC and black hair color (BHC) individuals. We also compared gene expression in melanoma tumors from individuals with RHC versus BHC. Gene expression in normal skin from RHC cutaneous cells showed dysregulation in 8 out of 23 hub genes (CLN3, ATG10, WIPI2, SNX2, GABARAPL2, YWHA, PCNA and GBAS). Hub genes did not differ between melanoma tumors in RHC versus BHC individuals. The study suggests that healthy skin cells from RHC individuals present a constitutive genomic deregulation associated with the red hair phenotype and identify novel genes involved in melanocyte biology.

  4. A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells

    PubMed Central

    Berman, Jennifer R.; Postovit, Lynne-Marie

    2016-01-01

    The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation. PMID:27089539

  5. Efficient Synergistic Single-Cell Genome Assembly.

    PubMed

    Movahedi, Narjes S; Embree, Mallory; Nagarajan, Harish; Zengler, Karsten; Chitsaz, Hamidreza

    2016-01-01

    As the vast majority of all microbes are unculturable, single-cell sequencing has become a significant method to gain insight into microbial physiology. Single-cell sequencing methods, currently powered by multiple displacement genome amplification (MDA), have passed important milestones such as finishing and closing the genome of a prokaryote. However, the quality and reliability of genome assemblies from single cells are still unsatisfactory due to uneven coverage depth and the absence of scattered chunks of the genome in the final collection of reads caused by MDA bias. In this work, our new algorithm Hybrid De novo Assembler (HyDA) demonstrates the power of coassembly of multiple single-cell genomic data sets through significant improvement of the assembly quality in terms of predicted functional elements and length statistics. Coassemblies contain significantly more base pairs and protein coding genes, cover more subsystems, and consist of longer contigs compared to individual assemblies by the same algorithm as well as state-of-the-art single-cell assemblers SPAdes and IDBA-UD. Hybrid De novo Assembler (HyDA) is also able to avoid chimeric assemblies by detecting and separating shared and exclusive pieces of sequence for input data sets. By replacing one deep single-cell sequencing experiment with a few single-cell sequencing experiments of lower depth, the coassembly method can hedge against the risk of failure and loss of the sample, without significantly increasing sequencing cost. Application of the single-cell coassembler HyDA to the study of three uncultured members of an alkane-degrading methanogenic community validated the usefulness of the coassembly concept. HyDA is open source and publicly available at http://chitsazlab.org/software.html, and the raw reads are available at http://chitsazlab.org/research.html.

  6. TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol

    PubMed Central

    Picher, Ángel J.; Budeus, Bettina; Wafzig, Oliver; Krüger, Carola; García-Gómez, Sara; Martínez-Jiménez, María I.; Díaz-Talavera, Alberto; Weber, Daniela; Blanco, Luis; Schneider, Armin

    2016-01-01

    Sequencing of a single-cell genome requires DNA amplification, a process prone to introducing bias and errors into the amplified genome. Here we introduce a novel multiple displacement amplification (MDA) method based on the unique DNA primase features of Thermus thermophilus (Tth) PrimPol. TthPrimPol displays a potent primase activity preferring dNTPs as substrates unlike conventional primases. A combination of TthPrimPol's unique ability to synthesize DNA primers with the highly processive Phi29 DNA polymerase (Φ29DNApol) enables near-complete whole genome amplification from single cells. This novel method demonstrates superior breadth and evenness of genome coverage, high reproducibility, excellent single-nucleotide variant (SNV) detection rates with low allelic dropout (ADO) and low chimera formation as exemplified by sequencing HEK293 cells. Moreover, copy number variant (CNV) calling yields superior results compared with random primer-based MDA methods. The advantages of this method, which we named TruePrime, promise to facilitate and improve single-cell genomic analysis. PMID:27897270

  7. Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms

    PubMed Central

    Zhang, Qiang; Wang, Tingting; Zhou, Qian; Zhang, Peng; Gong, Yanhai; Gou, Honglei; Xu, Jian; Ma, Bo

    2017-01-01

    Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches. PMID:28112223

  8. Phylogeography, Salinity Adaptations and Metabolic Potential of the Candidate Division KB1 Bacteria Based on a Partial Single Cell Genome

    PubMed Central

    Nigro, Lisa M.; Hyde, Andrew S.; MacGregor, Barbara J.; Teske, Andreas

    2016-01-01

    Deep-sea hypersaline anoxic basins and other hypersaline environments contain abundant and diverse microbial life that has adapted to these extreme conditions. The bacterial Candidate Division KB1 represents one of several uncultured groups that have been consistently observed in hypersaline microbial diversity studies. Here we report the phylogeography of KB1, its phylogenetic relationships to Candidate Division OP1 Bacteria, and its potential metabolic and osmotic stress adaptations based on a partial single cell amplified genome of KB1 from Orca Basin, the largest hypersaline seafloor brine basin in the Gulf of Mexico. Our results are consistent with the hypothesis – previously developed based on 14C incorporation experiments with mixed-species enrichments from Mediterranean seafloor brines – that KB1 has adapted its proteins to elevated intracellular salinity, but at the same time KB1 apparently imports glycine betaine; this compatible solute is potentially not limited to osmoregulation but could also serve as a carbon and energy source. PMID:27597842

  9. Genomic DNA of MCF-7 breast cancer cells not an ideal choice as positive control for PCR amplification based detection of Mouse Mammary Tumor Virus-Like Sequences.

    PubMed

    Kulkarni, Bhushan B; Hiremath, Shivaprakash V; Kulkarni, Suyamindra S; Hallikeri, Umesh R; Patil, Basavaraj R; Gai, Pramod B

    2013-11-01

    The identification of the etiology of breast cancer is a crucial research issue for the development of an effective preventive and treatment strategies. Researchers are exploring the possible involvement of Mouse Mammary Tumor Virus (MMTV) in causing human breast cancer. Hence, it becomes very important to use a consistent positive control agent in PCR amplification based detection of MMTV-Like Sequence (MMTV-LS) in human breast cancer for accurate and reproducible results. This study was done to investigate the feasibility of using genomic DNA of MCF-7 breast cancer cells to detect MMTV-LS using PCR amplification based detection. MMTV env and SAG gene located at the 3' long terminal repeat (LTR) sequences were targeted for the PCR based detection. No amplification was observed in case of the genomic DNA of MCF-7 breast cancer cells. However, the 2.7 kb DNA fragment comprising MMTV env and SAG LTR sequences yielded the products of desired size. From these results it can be concluded that Genomic DNA of MCF-7 cell is not a suitable choice as positive control for PCR or RT-PCR based detection of MMTV-LS. It is also suggested that plasmids containing the cloned genes or sequences of MMTV be used as positive control for detection of MMTV-LS.

  10. Identifying anti-cancer drug response related genes using an integrative analysis of transcriptomic and genomic variations with cell line-based drug perturbations

    PubMed Central

    Chen, Yunqin; Ma, Qin; Wei, Jia; Liu, Qi

    2016-01-01

    Background Clinical responses to anti-cancer therapies often only benefit a defined subset of patients. Predicting the best treatment strategy hinges on our ability to effectively translate genomic data into actionable information on drug responses. Results To achieve this goal, we compiled a comprehensive collection of baseline cancer genome data and drug response information derived from a large panel of cancer cell lines. This data set was applied to identify the signature genes relevant to drug sensitivity and their resistance by integrating CNVs and the gene expression of cell lines with in vitro drug responses. We presented an efficient in-silico pipeline for integrating heterogeneous cell line data sources with the simultaneous modeling of drug response values across all the drugs and cell lines. Potential signature genes correlated with drug response (sensitive or resistant) in different cancer types were identified. Using signature genes, our collaborative filtering-based drug response prediction model outperformed the 44 algorithms submitted to the DREAM competition on breast cancer cells. The functions of the identified drug response related signature genes were carefully analyzed at the pathway level and the synthetic lethality level. Furthermore, we validated these signature genes by applying them to the classification of the different subtypes of the TCGA tumor samples, and further uncovered their in vivo implications using clinical patient data. Conclusions Our work may have promise in translating genomic data into customized marker genes relevant to the response of specific drugs for a specific cancer type of individual patients. PMID:26824188

  11. Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

    PubMed

    Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2015-09-15

    Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.

  12. Genome regulation in mammalian cells.

    PubMed

    Puck, T T; Krystosek, A; Chan, D C

    1990-05-01

    A theory is presented proposing that genetic regulation in mammalian cells is at least a two-tiered effect; that one level of regulation involves the transition between gene exposure and sequestration; that normal differentiation requires a different spectrum of genes to be exposed in each separate state of differentiation; that the fiber systems of the cell cytoskeleton and the nuclear matrix together control the degree of gene exposure; that specific phosphorylation of these elements causes them to assume a different organizational network and to impose a different pattern of sequestration and exposure on the elements of the genome; that the varied gene phosphorylation mechanisms in the cell are integrated in this function; that attachment of this network system to specific parts of the chromosomes brings about sequestration or exposure of the genes in their neighborhood in a fashion similar to that observed when microtubule elements attach through the kinetochore to the centromeric DNA; that one function of repetitive sequences is to serve as elements for the final attachment of this fibrous network to the specific chromosomal loci; and that at least an important part of the calcium manifestation as a metabolic trigger of different differentiation states involves its acting as a binding agent to centers of electronegativity, in particular proteins and especially phosphorylated groups, so as to change the conformation of the fiber network that ultimately controls gene exposure in the mammalian cell. It would appear essential to determine what abnormal gene exposures and sequestrations are characteristic of each type of cancer; which agonists, if any, will bring about reverse transformation; and whether these considerations can be used in therapy.

  13. Inferring Cell Differentiation Processes Based on Phylogenetic Analysis of Genome-Wide Epigenetic Information: Hematopoiesis as a Model Case

    PubMed Central

    Koyanagi, Kanako O.

    2015-01-01

    How cells divide and differentiate is a fundamental question in organismal development; however, the discovery of differentiation processes in various cell types is laborious and sometimes impossible. Phylogenetic analysis is typically used to reconstruct evolutionary processes based on inherent characters. It could also be used to reconstruct developmental processes based on the developmental changes that occur during cell proliferation and differentiation. In this study, DNA methylation information from differentiated hematopoietic cells was used to perform phylogenetic analyses. The results were assessed for their validity in inferring hierarchical differentiation processes of hematopoietic cells and DNA methylation processes of differentiating progenitor cells. Overall, phylogenetic analyses based on DNA methylation information facilitated inferences regarding hematopoiesis. PMID:25638259

  14. Development and Validation of a Gene-Based Model for Outcome Prediction in Germ Cell Tumors Using a Combined Genomic and Expression Profiling Approach.

    PubMed

    Korkola, James E; Heck, Sandy; Olshen, Adam B; Feldman, Darren R; Reuter, Victor E; Houldsworth, Jane; Bosl, George J; Chaganti, R S K

    2015-01-01

    Germ Cell Tumors (GCT) have a high cure rate, but we currently lack the ability to accurately identify the small subset of patients who will die from their disease. We used a combined genomic and expression profiling approach to identify genomic regions and underlying genes that are predictive of outcome in GCT patients. We performed array-based comparative genomic hybridization (CGH) on 53 non-seminomatous GCTs (NSGCTs) treated with cisplatin based chemotherapy and defined altered genomic regions using Circular Binary Segmentation. We identified 14 regions associated with two year disease-free survival (2yDFS) and 16 regions associated with five year disease-specific survival (5yDSS). From corresponding expression data, we identified 101 probe sets that showed significant changes in expression. We built several models based on these differentially expressed genes, then tested them in an independent validation set of 54 NSGCTs. These predictive models correctly classified outcome in 64-79.6% of patients in the validation set, depending on the endpoint utilized. Survival analysis demonstrated a significant separation of patients with good versus poor predicted outcome when using a combined gene set model. Multivariate analysis using clinical risk classification with the combined gene model indicated that they were independent prognostic markers. This novel set of predictive genes from altered genomic regions is almost entirely independent of our previously identified set of predictive genes for patients with NSGCTs. These genes may aid in the identification of the small subset of patients who are at high risk of poor outcome.

  15. Genome Editing Using Mammalian Haploid Cells

    PubMed Central

    Horii, Takuro; Hatada, Izuho

    2015-01-01

    Haploid cells are useful for studying gene functions because disruption of a single allele can cause loss-of-function phenotypes. Recent success in generating haploid embryonic stem cells (ESCs) in mice, rats, and monkeys provides a new platform for simple genetic manipulation of the mammalian genome. Use of haploid ESCs enhances the genome-editing potential of the CRISPR/Cas system. For example, CRISPR/Cas was used in haploid ESCs to generate multiple knockouts and large deletions at high efficiency. In addition, genome-wide screening is facilitated by haploid cell lines containing gene knockout libraries. PMID:26437403

  16. Role of the mitochondrial genome in assisted reproductive technologies and embryonic stem cell-based therapeutic cloning.

    PubMed

    Brenner, Carol A; Kubisch, H Michael; Pierce, Kenneth E

    2004-01-01

    Mitochondria play a pivotal role in cellular metabolism and are important determinants of embryonic development. Mitochondrial function and biogenesis rely on an intricate coordination of regulation and expression of nuclear and mitochondrial genes. For example, several nucleus-derived transcription factors, such as mitochondrial transcription factor A, are required for mitochondrial DNA replication. Mitochondrial inheritance is strictly maternal while paternally-derived mitochondria are selectively eliminated during early embryonic cell divisions. However, there are reports from animals as well as human patients that paternal mitochondria can occasionally escape elimination, which in some cases has led to severe pathologies. The resulting existence of different mitochondrial genomes within the same cell has been termed mitochondrial heteroplasmy. The increasing use of invasive techniques in assisted reproduction in humans has raised concerns that one of the outcomes of such techniques is an increase in the incidence of mitochondrial heteroplasmy. Indeed, there is evidence that heteroplasmy is a direct consequence of ooplasm transfer, a technique that was used to 'rescue' oocytes from older women by injecting ooplasm from young oocytes. Mitochondria from donor and recipient were found in varying proportions in resulting children. Heteroplasmy is also a byproduct of nuclear transfer, as has been shown in studies on cloned sheep, cattle and monkeys. As therapeutic cloning will depend on nuclear transfer into oocytes and the subsequent generation of embryonic stem cells from resulting blastocysts, the prospect of mitochondrial heteroplasmy and its potential problems necessitate further studies in this area.

  17. Single Cell Genomics: Advances and Future Perspectives

    PubMed Central

    Macaulay, Iain C.; Voet, Thierry

    2014-01-01

    Advances in whole-genome and whole-transcriptome amplification have permitted the sequencing of the minute amounts of DNA and RNA present in a single cell, offering a window into the extent and nature of genomic and transcriptomic heterogeneity which occurs in both normal development and disease. Single-cell approaches stand poised to revolutionise our capacity to understand the scale of genomic, epigenomic, and transcriptomic diversity that occurs during the lifetime of an individual organism. Here, we review the major technological and biological breakthroughs achieved, describe the remaining challenges to overcome, and provide a glimpse into the promise of recent and future developments. PMID:24497842

  18. Human genome project and sickle cell disease.

    PubMed

    Norman, Brenda J; Miller, Sheila D

    2011-01-01

    Sickle cell disease is one of the most common genetic blood disorders in the United States that affects 1 in every 375 African Americans. Sickle cell disease is an inherited condition caused by abnormal hemoglobin in the red blood cells. The Human Genome Project has provided valuable insight and extensive research advances in the understanding of the human genome and sickle cell disease. Significant progress in genetic knowledge has led to an increase in the ability for researchers to map and sequence genes for diagnosis, treatment, and prevention of sickle cell disease and other chronic illnesses. This article explores some of the recent knowledge and advances about sickle cell disease and the Human Genome Project.

  19. Automated cell analysis tool for a genome-wide RNAi screen with support vector machine based supervised learning

    NASA Astrophysics Data System (ADS)

    Remmele, Steffen; Ritzerfeld, Julia; Nickel, Walter; Hesser, Jürgen

    2011-03-01

    RNAi-based high-throughput microscopy screens have become an important tool in biological sciences in order to decrypt mostly unknown biological functions of human genes. However, manual analysis is impossible for such screens since the amount of image data sets can often be in the hundred thousands. Reliable automated tools are thus required to analyse the fluorescence microscopy image data sets usually containing two or more reaction channels. The herein presented image analysis tool is designed to analyse an RNAi screen investigating the intracellular trafficking and targeting of acylated Src kinases. In this specific screen, a data set consists of three reaction channels and the investigated cells can appear in different phenotypes. The main issue of the image processing task is an automatic cell segmentation which has to be robust and accurate for all different phenotypes and a successive phenotype classification. The cell segmentation is done in two steps by segmenting the cell nuclei first and then using a classifier-enhanced region growing on basis of the cell nuclei to segment the cells. The classification of the cells is realized by a support vector machine which has to be trained manually using supervised learning. Furthermore, the tool is brightness invariant allowing different staining quality and it provides a quality control that copes with typical defects during preparation and acquisition. A first version of the tool has already been successfully applied for an RNAi-screen containing three hundred thousand image data sets and the SVM extended version is designed for additional screens.

  20. High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system.

    PubMed

    Endo, Yaeta; Sawasaki, Tatsuya

    2004-01-01

    Current cell-free protein expression systems are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. Escherichia coli based systems are not always sufficient for expression of eukaryotic proteins. This report reviews a high-throughput protein production method based on the cell-free system prepared from eukaryote, wheat embryos. We first demonstrate a method for preparation of this extract that exhibited a high degree of stability and activity. To maximize translation yield and throughput, we address and resolve the following issues: (1) optimization of the ORF flanking regions; (2) PCR-based generation of DNA for mRNA production; (3) expression vectors for large-scale protein production; and (4) a translation reaction that does not require a membrane. The combination of these elemental processes with robotic automation resulted in high-throughput protein synthesis.

  1. BrucellaBase: Genome information resource.

    PubMed

    Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Khader, L K M Abdul; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2016-09-01

    Brucella sp. causes a major zoonotic disease, brucellosis. Brucella belongs to the family Brucellaceae under the order Rhizobiales of Alphaproteobacteria. We present BrucellaBase, a web-based platform, providing features of a genome database together with unique analysis tools. We have developed a web version of the multilocus sequence typing (MLST) (Whatmore et al., 2007) and phylogenetic analysis of Brucella spp. BrucellaBase currently contains genome data of 510 Brucella strains along with the user interfaces for BLAST, VFDB, CARD, pairwise genome alignment and MLST typing. Availability of these tools will enable the researchers interested in Brucella to get meaningful information from Brucella genome sequences. BrucellaBase will regularly be updated with new genome sequences, new features along with improvements in genome annotations. BrucellaBase is available online at http://www.dbtbrucellosis.in/brucellabase.html or http://59.99.226.203/brucellabase/homepage.html.

  2. Fish T cells: recent advances through genomics

    USGS Publications Warehouse

    Laing, Kerry J.; Hansen, John D.

    2011-01-01

    This brief review is intended to provide a concise overview of the current literature concerning T cells, advances in identifying distinct T cell functional subsets, and in distinguishing effector cells from memory cells. We compare and contrast a wealth of recent progress made in T cell immunology of teleost, elasmobranch, and agnathan fish, to knowledge derived from mammalian T cell studies. From genome studies, fish clearly have most components associated with T cell function and we can speculate on the presence of putative T cell subsets, and the ability to detect their differentiation to form memory cells. Some recombinant proteins for T cell associated cytokines and antibodies for T cell surface receptors have been generated that will facilitate studying the functional roles of teleost T cells during immune responses. Although there is still a long way to go, major advances have occurred in recent years for investigating T cell responses, thus phenotypic and functional characterization is on the near horizon.

  3. Genome-editing Technologies for Gene and Cell Therapy.

    PubMed

    Maeder, Morgan L; Gersbach, Charles A

    2016-03-01

    Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed.

  4. Genome-editing Technologies for Gene and Cell Therapy

    PubMed Central

    Maeder, Morgan L; Gersbach, Charles A

    2016-01-01

    Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed. PMID:26755333

  5. Copy Number Variation Analysis by Array Analysis of Single Cells Following Whole Genome Amplification.

    PubMed

    Dimitriadou, Eftychia; Zamani Esteki, Masoud; Vermeesch, Joris Robert

    2015-01-01

    Whole genome amplification is required to ensure the availability of sufficient material for copy number variation analysis of a genome deriving from an individual cell. Here, we describe the protocols we use for copy number variation analysis of non-fixed single cells by array-based approaches following single-cell isolation and whole genome amplification. We are focusing on two alternative protocols, an isothermal and a PCR-based whole genome amplification method, followed by either comparative genome hybridization (aCGH) or SNP array analysis, respectively.

  6. Covering Your Bases: Inheritance of DNA Methylation in Plant Genomes

    PubMed Central

    Schmitz, Robert J.

    2014-01-01

    Cytosine methylation is an important base modification that is inherited across mitotic and meiotic cell divisions in plant genomes. Heritable methylation variants can contribute to within-species phenotypic variation. Few methylation variants were known until recently, making it possible to begin to address major unanswered questions: the extent of natural methylation variation within plant genomes, its effects on phenotypic variation, its degree of dependence on genotype, and how it fits into an evolutionary context. Techniques like whole-genome bisulfite sequencing (WGBS) make it possible to determine cytosine methylation states at single-base resolution across entire genomes and populations. Application of this method to natural and novel experimental populations is revealing answers to these long-standing questions about the role of DNA methylation in plant genomes. PMID:24270503

  7. Radiation induced genomic instability in bystander cells

    NASA Astrophysics Data System (ADS)

    Zhou, H.; Gu, S.; Randers-Pehrson, G.; Hei, T.

    There is considerable evidence that exposure to ionizing radiation may induce a heritable genomic instability that leads to a persisting increased frequency of genetic and functional changes in the non-irradiated progeny of a wide variety of irradiated cells Genomic instability is measured as delayed expressions in chromosomal alterations micronucleus formation gene mutations and decreased plating efficiency During the last decade numerous studies have shown that radiation could induce bystander effect in non-irradiated neighboring cells similar endpoints have also been used in genomic instability studies Both genomic instability and the bystander effect are phenomena that result in a paradigm shift in our understanding of radiation biology In the past it seemed reasonable to assume that the production of single- and double-strand DNA breaks are due to direct energy deposition of energy by a charged particle to the nucleus It turns out that biology is not quite that simple Using the Columbia University charged particle microbeam and the highly sensitive human hamster hybrid AL cell mutagenic assay we irradiated 10 of the cells with a lethal dose of 30 alpha particles through the nucleus After overnight incubation the remaining viable bystander cells were replated in dishes for colony formation Clonal isolates were expanded and cultured for 6 consecutive weeks to assess plating efficiency and mutation frequency Preliminary results indicated that there was no significant decrease in plating efficiency among the bystander colonies when compared with

  8. Genomic Landscape of Developing Male Germ Cells

    PubMed Central

    Lee, Tin-Lap; Pang, Alan Lap-Yin; Rennert, Owen M.; Chan, Wai-Yee

    2010-01-01

    Spermatogenesis is a highly orchestrated developmental process by which spermatogonia develop into mature spermatozoa. This process involves many testis- or male germ cell-specific gene products whose expressions are strictly regulated. In the past decade the advent of high-throughput gene expression analytical techniques has made functional genomic studies of this process, particularly in model animals such as mice and rats, feasible and practical. These studies have just begun to reveal the complexity of the genomic landscape of the developing male germ cells. Over 50% of the mouse and rat genome are expressed during testicular development. Among transcripts present in germ cells, 40% – 60% are uncharacterized. A number of genes, and consequently their associated biological pathways, are differentially expressed at different stages of spermatogenesis. Developing male germ cells present a rich repertoire of genetic processes. Tissue-specific as well as spermatogenesis stage-specific alternative splicing of genes exemplifies the complexity of genome expression. In addition to this layer of control, discoveries of abundant presence of antisense transcripts, expressed psuedogenes, non-coding RNAs (ncRNA) including long ncRNAs, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), and retrogenes all point to the presence of multiple layers of expression and functional regulation in male germ cells. It is anticipated that application of systems biology approaches will further our understanding of the regulatory mechanism of spermatogenesis.† PMID:19306351

  9. Whole Genome Amplification of Labeled Viable Single Cells Suited for Array-Comparative Genomic Hybridization.

    PubMed

    Kroneis, Thomas; El-Heliebi, Amin

    2015-01-01

    Understanding details of a complex biological system makes it necessary to dismantle it down to its components. Immunostaining techniques allow identification of several distinct cell types thereby giving an inside view of intercellular heterogeneity. Often staining reveals that the most remarkable cells are the rarest. To further characterize the target cells on a molecular level, single cell techniques are necessary. Here, we describe the immunostaining, micromanipulation, and whole genome amplification of single cells for the purpose of genomic characterization. First, we exemplify the preparation of cell suspensions from cultured cells as well as the isolation of peripheral mononucleated cells from blood. The target cell population is then subjected to immunostaining. After cytocentrifugation target cells are isolated by micromanipulation and forwarded to whole genome amplification. For whole genome amplification, we use GenomePlex(®) technology allowing downstream genomic analysis such as array-comparative genomic hybridization.

  10. DNA damage in cells exhibiting radiation-induced genomic instability

    DOE PAGES

    Keszenman, Deborah J.; Kolodiuk, Lucia; Baulch, Janet E.

    2015-02-22

    Cells exhibiting radiation induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during normal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesismore » that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.« less

  11. DNA damage in cells exhibiting radiation-induced genomic instability

    SciTech Connect

    Keszenman, Deborah J.; Kolodiuk, Lucia; Baulch, Janet E.

    2015-02-22

    Cells exhibiting radiation induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during normal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesis that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.

  12. Genome-editing tools for stem cell biology

    PubMed Central

    Vasileva, E A; Shuvalov, O U; Garabadgiu, A V; Melino, G; Barlev, N A

    2015-01-01

    Human pluripotent stem cells provide a versatile platform for regenerative studies, drug testing and disease modeling. That the expression of only four transcription factors, Oct4, Klf4, Sox2 and c-Myc (OKSM), is sufficient for generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells has revolutionized the field and also highlighted the importance of OKSM as targets for genome editing. A number of novel genome-editing systems have been developed recently. In this review, we focus on successful applications of several such systems for generation of iPSCs. In particular, we discuss genome-editing systems based on zinc-finger fusion proteins (ZFs), transcription activator-like effectors (TALEs) and an RNA-guided DNA-specific nuclease, Cas9, derived from the bacterial defense system against viruses that utilizes clustered regularly interspaced short palindromic repeats (CRISPR). PMID:26203860

  13. Genome Annotation in a Community College Cell Biology Lab

    ERIC Educational Resources Information Center

    Beagley, C. Timothy

    2013-01-01

    The Biology Department at Salt Lake Community College has used the IMG-ACT toolbox to introduce a genome mapping and annotation exercise into the laboratory portion of its Cell Biology course. This project provides students with an authentic inquiry-based learning experience while introducing them to computational biology and contemporary learning…

  14. Synthetic genomics and the construction of a synthetic bacterial cell.

    PubMed

    Glass, John I

    2012-01-01

    The first synthetic cellular organism was created in 2010 and based on a very small, very simple bacterium called Mycoplasma mycoides. The bacterium was called synthetic because its DNA genome was chemically synthesized rather than replicated from an existing template DNA, as occurs in all other known cellular life on Earth. The experiment was undertaken in order to develop a system that would allow creation of a minimal bacterial cell that could lead to a better understand of the first principles of cellular life. The effort resulted in new synthetic genomics techniques called genome assembly and genome transplantation. The ability of scientists to design and build bacteria opens new possibilities for creating microbes to solve human problems.

  15. Dissecting genomic diversity, one cell at a time

    PubMed Central

    Blainey, Paul C; Quake, Stephen R

    2014-01-01

    Emerging technologies are bringing single-cell genome sequencing into the mainstream; this field has already yielded insights into the genetic architecture and variability between cells that highlight the dynamic nature of the genome. PMID:24524132

  16. Genome integrity, stem cells and hyaluronan

    PubMed Central

    Darzynkiewicz, Zbigniew; Balazs, Endre A.

    2012-01-01

    Faithful preservation of genome integrity is the critical mission of stem cells as well as of germ cells. Reviewed are the following mechanisms involved in protecting DNA in these cells: (a) The efflux machinery that can pump out variety of genotoxins in ATP-dependent manner; (b) the mechanisms maintaining minimal metabolic activity which reduces generation of reactive oxidants, by-products of aerobic respiration; (c) the role of hypoxic niche of stem cells providing a gradient of variable oxygen tension; (d) (e) the presence of hyaluronan (HA) and HA receptors on stem cells and in the niche; (f) the role of HA in protecting DNA from oxidative damage; (g) the specific function of HA in protecting DNA in stem cells; (h) the interactions of HA with sperm cells and oocytes that also may shield their DNA from oxidative damage, and (e) mechanisms by which HA exerts the anti-oxidant activity. While HA has multitude of functions its anti-oxidant capabilities are often overlooked but may be of significance in preservation of integrity of stem and germ cells genome. PMID:22383371

  17. Genomics and epigenomics of renal cell carcinoma.

    PubMed

    Maher, Eamonn R

    2013-02-01

    Kidney cancer accounts for about 2% of all cancers and worldwide >250,000 new cases of kidney cancer are diagnosed each year. Renal cell carcinoma (RCC) is the most common form of adult kidney cancer and this review describes our current knowledge of the genetic and epigenetic basis of sporadic RCC. Though to date major advances in understanding the underlying the molecular basis of renal cell carcinoma (RCC) have often been derived from studies of rare familial forms of renal cell carcinoma, large-scale genomic and epigenomic studies of sporadic tumours are beginning to provide clearer pictures of the genomic and epigenomic landscape of RCC and the key pathways implicated in the initiation and progression of the disease. Although current knowledge of the molecular pathogenesis of RCC is incomplete, and mostly relates to clear cell (conventional) RCC, the next five years will see an unprecedented flood of genomic and epigenomic data and the key future challenges will relate to the utilisation of this data to develop novel genetic and epigenetic markers for diagnosis and prognosis and to develop novel targeted therapies in order to enable an age of personalised medicine.

  18. Microfluidic whole genome amplification device for single cell sequencing.

    PubMed

    Yu, Zhilong; Lu, Sijia; Huang, Yanyi

    2014-10-07

    We developed a microfluidic device to perform multiplex single-cell whole-genome amplification (WGA) using multiple annealing and looping-based amplification cycles (MALBAC). This device, made of polydimethylsiloxane (PDMS), allows us to monitor the whole process of cell loading and single-cell WGA for sequencing. We show that the genome coverage of MALBAC amplifications is reproducible between chambers on a single chip and between different chips, which enables data normalization using standard samples to accurately identify copy number variations (CNVs). This device provides an easy-to-operate approach to perform single cell sequencing library preparation with minimum hands-on time. It reduces the requirement of manual expertise as well as the risk of contamination, which is essential in future applications especially the medical diagnosis.

  19. Genome-Wide Association Study of Prognosis in Advanced Non–Small Cell Lung Cancer Patients Receiving Platinum-Based Chemotherapy

    PubMed Central

    Hu, Lingmin; Wu, Chen; Zhao, Xueying; Heist, Rebecca; Su, Li; Zhao, Yang; Han, Baohui; Cao, Songyu; Chu, Minjie; Dai, Juncheng; Dong, Jing; Shu, Yongqian; Xu, Lin; Chen, Yijiang; Wang, Yi; Lu, Feng; Jiang, Yue; Yu, Dianke; Chen, Hongyan; Tan, Wen; Ma, Hongxia; Chen, Jiaping; Jin, Guangfu; Wu, Tangchun; Lu, Daru; Christiani, David C.; Lin, Dongxin; Hu, Zhibin; Shen, Hongbing

    2013-01-01

    Purpose Genetic variation may influence chemotherapy response and overall survival in cancer patients. Experimental design We conducted a genome-wide scan in 535 advanced-stage non–small cell lung cancer (NSCLC) patients from two independent cohorts (307 from Nanjing and 228 from Beijing). A replication was carried out on an independent cohort of 340 patients from Southeastern China followed by a second validation on 409 patients from the Massachusetts General Hospital (Boston, MA). Results Consistent associations with NSCLC survival were identified for five single-nucleotide polymorphisms (SNP) in Chinese populations with P values ranging from 3.63 × 10−5 to 4.19 × 10−7 in the additive genetic model. The minor allele of three SNPs (rs7629386 at 3p22.1, rs969088 at 5p14.1, and rs3850370 at 14q24.3) were associated with worse NSCLC survival while 2 (rs41997 at 7q31.31 and rs12000445 at 9p21.3) were associated with better NSCLC survival. In addition, rs7629386 at 3p22.1 (CTNNB1) and rs3850370 at 14q24.3 (SNW1-ALKBH1-NRXN3) were further replicated in the Caucasian population. Conclusion In this three-stage genome-wide association studies, we identified five SNPs as markers for survival of advanced-stage NSCLC patients treated with first-line platinum-based chemotherapy in Chinese Han populations. Two of these SNPs, rs7629386 and rs3850370, could also be markers for survival among Caucasian patients. PMID:22872573

  20. Population-based 3D genome structure analysis reveals driving forces in spatial genome organization

    PubMed Central

    Li, Wenyuan; Kalhor, Reza; Dai, Chao; Hao, Shengli; Gong, Ke; Zhou, Yonggang; Li, Haochen; Zhou, Xianghong Jasmine; Le Gros, Mark A.; Larabell, Carolyn A.; Chen, Lin; Alber, Frank

    2016-01-01

    Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization. PMID:26951677

  1. Selective single cell isolation for genomics using microraft arrays

    PubMed Central

    Welch, Joshua D.; Williams, Lindsay A.; DiSalvo, Matthew; Brandt, Alicia T.; Marayati, Raoud; Sims, Christopher E.; Allbritton, Nancy L.; Prins, Jan F.; Yeh, Jen Jen; Jones, Corbin D.

    2016-01-01

    Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance. PMID:27530426

  2. Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

    PubMed

    Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

    2015-01-01

    We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

  3. Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells.

    PubMed

    Ju, Young Seok; Tubio, Jose M C; Mifsud, William; Fu, Beiyuan; Davies, Helen R; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J; Tan, Benita K T; Aparicio, Samuel; Span, Paul N; Martens, John W M; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Myklebost, Ola; Flanagan, Adrienne M; Foster, Christopher; Neal, David E; Cooper, Colin; Eeles, Rosalind; Bova, Steven G; Lakhani, Sunil R; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L; Purdie, Colin A; Thompson, Alastair M; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J; Stratton, Michael R

    2015-06-01

    Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells.

  4. The somatic genomic landscape of chromophobe renal cell carcinoma

    PubMed Central

    Davis, Caleb F.; Ricketts, Christopher; Wang, Min; Yang, Lixing; Cherniack, Andrew D.; Shen, Hui; Buhay, Christian; Kang, Hyojin; Kim, Sang Cheol; Fahey, Catherine C.; Hacker, Kathryn E.; Bhanot, Gyan; Gordenin, Dmitry A.; Chu, Andy; Gunaratne, Preethi H.; Biehl, Michael; Seth, Sahil; Kaipparettu, Benny A.; Bristow, Christopher A.; Donehower, Lawrence A.; Wallen, Eric M.; Smith, Angela B.; Tickoo, Satish K.; Tamboli, Pheroze; Reuter, Victor; Schmidt, Laura S.; Hsieh, James J.; Choueiri, Toni K.; Hakimi, A. Ari; Chin, Lynda; Meyerson, Matthew; Kucherlapati, Raju; Park, Woong-Yang; Robertson, A. Gordon; Laird, Peter W.; Henske, Elizabeth P.; Kwiatkowski, David J.; Park, Peter J.; Morgan, Margaret; Shuch, Brian; Muzny, Donna; Wheeler, David A.; Linehan, W. Marston; Gibbs, Richard A.; Rathmell, W. Kimryn; Creighton, Chad J.

    2014-01-01

    Summary We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) based on multidimensional and comprehensive characterization, including mitochondrial DNA (mtDNA) and whole genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared to other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT up-regulation in cancer distinct from previously-observed amplifications and point mutations. PMID:25155756

  5. From CO2 to cell: energetic expense of creating biomass using the Calvin-Benson-Bassham and reductive citric acid cycles based on genome data.

    PubMed

    Mangiapia, Mary; Scott, Kathleen

    2016-04-01

    The factors driving the dominance of the Calvin-Benson-Bassham cycle (CBB) or reductive citric acid cycle (rCAC) in autotrophic microorganisms in different habitats are debated. Based on costs for synthesizing a few metabolic intermediates, it has been suggested that the CBB poses a disadvantage due to higher metabolic cost. The purpose of this study was to extend this estimate of cost from metabolite synthesis to biomass synthesis. For 12 gammaproteobacteria (CBB) and five epsilonproteobacteria (rCAC), the amount of ATP to synthesize a gram of biomass from CO2 was calculated from genome sequences via metabolic maps. The eleven central carbon metabolites needed to synthesize biomass were all less expensive to synthesize via the rCAC (66%-89% of the ATP needed to synthesize them via CBB). Differences in cell compositions did result in differing demands for metabolites among the organisms, but the differences in cost to synthesize biomass were small among organisms that used a particular pathway (e.g. rCAC), compared to the difference between pathways (rCAC versus CBB). The rCAC autotrophs averaged 0.195 moles ATP per g biomass, while their CBB counterparts averaged 0.238. This is the first in silico estimate of the relative expense of both pathways to generate biomass.

  6. Stem cell genome-to-systems biology.

    PubMed

    Chia, Na-Yu; Ng, Huck-Hui

    2012-01-01

    Stem cells are capable of extended proliferation and concomitantly differentiating into a plethora of specialized cell types that render them apropos for their usage as a form of regenerative medicine for cell replacement therapies. The molecular processes that underlie the ability for stem cells to self-renew and differentiate have been intriguing, and elucidating the intricacies within the genome is pertinent to enhance our understanding of stem cells. Systems biology is emerging as a crucial field in the study of the sophisticated nature of stem cells, through the adoption of multidisciplinary approaches which couple high-throughput experimental techniques with computational and mathematical analysis. This allows for the determination of the molecular constituents that govern stem cell characteristics and conjointly with functional validations via genetic perturbation and protein location binding analysis necessitate the construction of the complex transcriptional regulatory network. With the elucidation of protein-protein interaction, protein-DNA regulation, microRNA involvement as well as the epigenetic modifications, it is possible to comprehend the defining features of stem cells at the system level.

  7. Whole-genome amplification of single-cell genomes for next-generation sequencing.

    PubMed

    Korfhage, Christian; Fisch, Evelyn; Fricke, Evelyn; Baedker, Silke; Loeffert, Dirk

    2013-10-11

    DNA sequence analysis and genotyping of biological samples using next-generation sequencing (NGS), microarrays, or real-time PCR is often limited by the small amount of sample available. A single cell contains only one to four copies of the genomic DNA, depending on the organism (haploid or diploid organism) and the cell-cycle phase. The DNA content of a single cell ranges from a few femtograms in bacteria to picograms in mammalia. In contrast, a deep analysis of the genome currently requires a few hundred nanograms up to micrograms of genomic DNA for library formation necessary for NGS sequencing or labeling protocols (e.g., microarrays). Consequently, accurate whole-genome amplification (WGA) of single-cell DNA is required for reliable genetic analysis (e.g., NGS) and is particularly important when genomic DNA is limited. The use of single-cell WGA has enabled the analysis of genomic heterogeneity of individual cells (e.g., somatic genomic variation in tumor cells). This unit describes how the genome of single cells can be used for WGA for further genomic studies, such as NGS. Recommendations for isolation of single cells are given and common sources of errors are discussed.

  8. [Research progress in developing reporter systems for the enrichment of positive cells with targeted genome modification].

    PubMed

    Bai, Yichun; Xu, Kun; Wei, Zehui; Ma, Zheng; Zhang, Zhiying

    2016-01-01

    Targeted genome editing technology plays an important role in studies of gene function, gene therapy and transgenic breeding. Moreover, the efficiency of targeted genome editing is increased dramatically with the application of recently developed artificial nucleases such as ZFNs, TALENs and CRISPR/Cas9. However, obtaining positive cells with targeted genome modification is restricted to some extent by nucleases expression plasmid transfection efficiency, nucleases expression and activity, and repair efficiency after genome editing. Thus, the enrichment and screening of positive cells with targeted genome modification remains a problem that need to be solved. Surrogate reporter systems could be used to reflect the efficiency of nucleases indirectly and enrich genetically modified positive cells effectively, which may increase the efficiency of the enrichment and screening of positive cells with targeted genome modification. In this review, we mainly summarized principles and applications of reporter systems based on NHEJ and SSA repair mechanisms, which may provide references for related studies in future.

  9. Evaluation of two public genome references for Chinese hamster ovary cells in the context of RNA-seq based gene expression analysis.

    PubMed

    Chen, Chun; Le, Huong; Goudar, Chetan T

    2017-03-15

    RNA-Seq is a powerful transcriptomics tool for mammalian cell culture process development. Successful RNA-Seq data analysis requires a high quality reference for read mapping and gene expression quantification. Currently, there are two public genome references for Chinese hamster ovary (CHO) cells, the predominant mammalian cell line in the biopharmaceutical industry. In this study, we compared these two references by analyzing 60 RNA-Seq samples from a variety of CHO cell culture conditions. Among the 20,891 common genes in both references, we observed that 31.5% have more than 7.1% quantification differences, implying gene definition differences in the two references. We propose a framework to quantify this difference using two metrics, Consistency and Stringency, which account for the average quantification difference between the two references over all samples, and the sample-specific effect on the quantification result, respectively. These two metrics can be used to identify potential genes for future gene model improvement and to understand the reliability of differentially expressed genes identified by RNA-Seq data analysis. Before a more comprehensive genome reference for CHO cells emerges, the strategy proposed in this study can enable a more robust transcriptome analysis for CHO cell RNA-Seq data. This article is protected by copyright. All rights reserved.

  10. Shared clonal cytogenetic abnormalities in aberrant mast cells and leukemic myeloid blasts detected by single nucleotide polymorphism microarray-based whole-genome scanning.

    PubMed

    Frederiksen, John K; Shao, Lina; Bixby, Dale L; Ross, Charles W

    2016-04-01

    Systemic mastocytosis (SM) is characterized by a clonal proliferation of aberrant mast cells within extracutaneous sites. In a subset of SM cases, a second associated hematologic non-mast cell disease (AHNMD) is also present, usually of myeloid origin. Polymerase chain reaction and targeted fluorescence in situ hybridization studies have provided evidence that, in at least some cases, the aberrant mast cells are related clonally to the neoplastic cells of the AHNMD. In this work, a single nucleotide polymorphism microarray (SNP-A) was used to characterize the cytogenetics of the aberrant mast cells from a patient with acute myeloid leukemia and concomitant mast cell leukemia associated with a KIT D816A mutation. The results demonstrate the presence of shared cytogenetic abnormalities between the mast cells and myeloid blasts, as well as additional abnormalities within mast cells (copy-neutral loss of heterozygosity) not detectable by routine karyotypic analysis. To our knowledge, this work represents the first application of SNP-A whole-genome scanning to the detection of shared cytogenetic abnormalities between the two components of a case of SM-AHNMD. The findings provide additional evidence of a frequent clonal link between aberrant mast cells and cells of myeloid AHNMDs, and also highlight the importance of direct sequencing for identifying uncommon activating KIT mutations.

  11. Defining cell types and states with single-cell genomics

    PubMed Central

    Trapnell, Cole

    2015-01-01

    A revolution in cellular measurement technology is under way: For the first time, we have the ability to monitor global gene regulation in thousands of individual cells in a single experiment. Such experiments will allow us to discover new cell types and states and trace their developmental origins. They overcome fundamental limitations inherent in measurements of bulk cell population that have frustrated efforts to resolve cellular states. Single-cell genomics and proteomics enable not only precise characterization of cell state, but also provide a stunningly high-resolution view of transitions between states. These measurements may finally make explicit the metaphor that C.H. Waddington posed nearly 60 years ago to explain cellular plasticity: Cells are residents of a vast “landscape” of possible states, over which they travel during development and in disease. Single-cell technology helps not only locate cells on this landscape, but illuminates the molecular mechanisms that shape the landscape itself. However, single-cell genomics is a field in its infancy, with many experimental and computational advances needed to fully realize its full potential. PMID:26430159

  12. Genome editing in pluripotent stem cells: research and therapeutic applications.

    PubMed

    Deleidi, Michela; Yu, Cong

    2016-05-06

    Recent progress in human pluripotent stem cell (hPSC) and genome editing technologies has opened up new avenues for the investigation of human biology in health and disease as well as the development of therapeutic applications. Gene editing approaches with programmable nucleases have been successfully established in hPSCs and applied to study gene function, develop novel animal models and perform genetic and chemical screens. Several studies now show the successful editing of disease-linked alleles in somatic and patient-derived induced pluripotent stem cells (iPSCs) as well as in animal models. Importantly, initial clinical trials have shown the safety of programmable nucleases for ex vivo somatic gene therapy. In this context, the unlimited proliferation potential and the pluripotent properties of iPSCs may offer advantages for gene targeting approaches. However, many technical and safety issues still need to be addressed before genome-edited iPSCs are translated into the clinical setting. Here, we provide an overview of the available genome editing systems and discuss opportunities and perspectives for their application in basic research and clinical practice, with a particular focus on hPSC based research and gene therapy approaches. Finally, we discuss recent research on human germline genome editing and its social and ethical implications.

  13. Effects of sample treatments on genome recovery via single-cell genomics

    SciTech Connect

    Clingenpeel, Scott; Schwientek, Patrick; Hugenholtz, Philip; Woyke, Tanja

    2014-06-13

    It is known that single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we demonstrate that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

  14. Genome-Based Taxonomic Classification of Bacteroidetes.

    PubMed

    Hahnke, Richard L; Meier-Kolthoff, Jan P; García-López, Marina; Mukherjee, Supratim; Huntemann, Marcel; Ivanova, Natalia N; Woyke, Tanja; Kyrpides, Nikos C; Klenk, Hans-Peter; Göker, Markus

    2016-01-01

    The bacterial phylum Bacteroidetes, characterized by a distinct gliding motility, occurs in a broad variety of ecosystems, habitats, life styles, and physiologies. Accordingly, taxonomic classification of the phylum, based on a limited number of features, proved difficult and controversial in the past, for example, when decisions were based on unresolved phylogenetic trees of the 16S rRNA gene sequence. Here we use a large collection of type-strain genomes from Bacteroidetes and closely related phyla for assessing their taxonomy based on the principles of phylogenetic classification and trees inferred from genome-scale data. No significant conflict between 16S rRNA gene and whole-genome phylogenetic analysis is found, whereas many but not all of the involved taxa are supported as monophyletic groups, particularly in the genome-scale trees. Phenotypic and phylogenomic features support the separation of Balneolaceae as new phylum Balneolaeota from Rhodothermaeota and of Saprospiraceae as new class Saprospiria from Chitinophagia. Epilithonimonas is nested within the older genus Chryseobacterium and without significant phenotypic differences; thus merging the two genera is proposed. Similarly, Vitellibacter is proposed to be included in Aequorivita. Flexibacter is confirmed as being heterogeneous and dissected, yielding six distinct genera. Hallella seregens is a later heterotypic synonym of Prevotella dentalis. Compared to values directly calculated from genome sequences, the G+C content mentioned in many species descriptions is too imprecise; moreover, corrected G+C content values have a significantly better fit to the phylogeny. Corresponding emendations of species descriptions are provided where necessary. Whereas most observed conflict with the current classification of Bacteroidetes is already visible in 16S rRNA gene trees, as expected whole-genome phylogenies are much better resolved.

  15. Genome-Based Taxonomic Classification of Bacteroidetes

    PubMed Central

    Hahnke, Richard L.; Meier-Kolthoff, Jan P.; García-López, Marina; Mukherjee, Supratim; Huntemann, Marcel; Ivanova, Natalia N.; Woyke, Tanja; Kyrpides, Nikos C.; Klenk, Hans-Peter; Göker, Markus

    2016-01-01

    The bacterial phylum Bacteroidetes, characterized by a distinct gliding motility, occurs in a broad variety of ecosystems, habitats, life styles, and physiologies. Accordingly, taxonomic classification of the phylum, based on a limited number of features, proved difficult and controversial in the past, for example, when decisions were based on unresolved phylogenetic trees of the 16S rRNA gene sequence. Here we use a large collection of type-strain genomes from Bacteroidetes and closely related phyla for assessing their taxonomy based on the principles of phylogenetic classification and trees inferred from genome-scale data. No significant conflict between 16S rRNA gene and whole-genome phylogenetic analysis is found, whereas many but not all of the involved taxa are supported as monophyletic groups, particularly in the genome-scale trees. Phenotypic and phylogenomic features support the separation of Balneolaceae as new phylum Balneolaeota from Rhodothermaeota and of Saprospiraceae as new class Saprospiria from Chitinophagia. Epilithonimonas is nested within the older genus Chryseobacterium and without significant phenotypic differences; thus merging the two genera is proposed. Similarly, Vitellibacter is proposed to be included in Aequorivita. Flexibacter is confirmed as being heterogeneous and dissected, yielding six distinct genera. Hallella seregens is a later heterotypic synonym of Prevotella dentalis. Compared to values directly calculated from genome sequences, the G+C content mentioned in many species descriptions is too imprecise; moreover, corrected G+C content values have a significantly better fit to the phylogeny. Corresponding emendations of species descriptions are provided where necessary. Whereas most observed conflict with the current classification of Bacteroidetes is already visible in 16S rRNA gene trees, as expected whole-genome phylogenies are much better resolved. PMID:28066339

  16. Are we Genomic Mosaics? Variations of the Genome of Somatic Cells can Contribute to Diversify our Phenotypes.

    PubMed

    Astolfi, P A; Salamini, F; Sgaramella, V

    2010-09-01

    Theoretical and experimental evidences support the hypothesis that the genomes and the epigenomes may be different in the somatic cells of complex organisms. In the genome, the differences range from single base substitutions to chromosome number; in the epigenome, they entail multiple postsynthetic modifications of the chromatin. Somatic genome variations (SGV) may accumulate during development in response both to genetic programs, which may differ from tissue to tissue, and to environmental stimuli, which are often undetected and generally irreproducible. SGV may jeopardize physiological cellular functions, but also create novel coding and regulatory sequences, to be exposed to intraorganismal Darwinian selection. Genomes acknowledged as comparatively poor in genes, such as humans', could thus increase their pristine informational endowment. A better understanding of SGV will contribute to basic issues such as the "nature vs nurture" dualism and the inheritance of acquired characters. On the applied side, they may explain the low yield of cloning via somatic cell nuclear transfer, provide clues to some of the problems associated with transdifferentiation, and interfere with individual DNA analysis. SGV may be unique in the different cells types and in the different developmental stages, and thus explain the several hundred gaps persisting in the human genomes "completed" so far. They may compound the variations associated to our epigenomes and make of each of us an "(epi)genomic" mosaic. An ensuing paradigm is the possibility that a single genome (the ephemeral one assembled at fertilization) has the capacity to generate several different brains in response to different environments.

  17. [CRISPR/Cas system for genome editing in pluripotent stem cells].

    PubMed

    Vasil'eva, E A; Melino, D; Barlev, N A

    2015-01-01

    Genome editing systems based on site-specific nucleases became very popular for genome editing in modern bioengineering. Human pluripotent stem cells provide a unique platform for genes function study, disease modeling, and drugs testing. Consequently, technology for fast, accurate and well controlled genome manipulation is required. CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated) system could be employed for these purposes. This system is based on site-specific programmable nuclease Cas9. Numerous advantages of the CRISPR/Cas system and its successful application to human stem cells provide wide opportunities for genome therapy and regeneration medicine. In this publication, we describe and compare the main genome editing systems based on site-specific programmable nucleases and discuss opportunities and perspectives of the CRISPR/Cas system for application to pluripotent stem cells.

  18. Accessing the genomic information of unculturable oceanic picoeukaryotes by combining multiple single cells

    PubMed Central

    Mangot, Jean-François; Logares, Ramiro; Sánchez, Pablo; Latorre, Fran; Seeleuthner, Yoann; Mondy, Samuel; Sieracki, Michael E.; Jaillon, Olivier; Wincker, Patrick; Vargas, Colomban de; Massana, Ramon

    2017-01-01

    Pico-sized eukaryotes play key roles in the functioning of marine ecosystems, but we still have a limited knowledge on their ecology and evolution. The MAST-4 lineage is of particular interest, since it is widespread in surface oceans, presents ecotypic differentiation and has defied culturing efforts so far. Single cell genomics (SCG) are promising tools to retrieve genomic information from these uncultured organisms. However, SCG are based on whole genome amplification, which normally introduces amplification biases that limit the amount of genomic data retrieved from a single cell. Here, we increase the recovery of genomic information from two MAST-4 lineages by co-assembling short reads from multiple Single Amplified Genomes (SAGs) belonging to evolutionary closely related cells. We found that complementary genomic information is retrieved from different SAGs, generating co-assembly that features >74% of genome recovery, against about 20% when assembled individually. Even though this approach is not aimed at generating high-quality draft genomes, it allows accessing to the genomic information of microbes that would otherwise remain unreachable. Since most of the picoeukaryotes still remain uncultured, our work serves as a proof-of-concept that can be applied to other taxa in order to extract genomic data and address new ecological and evolutionary questions. PMID:28128359

  19. Accessing the genomic information of unculturable oceanic picoeukaryotes by combining multiple single cells.

    PubMed

    Mangot, Jean-François; Logares, Ramiro; Sánchez, Pablo; Latorre, Fran; Seeleuthner, Yoann; Mondy, Samuel; Sieracki, Michael E; Jaillon, Olivier; Wincker, Patrick; Vargas, Colomban de; Massana, Ramon

    2017-01-27

    Pico-sized eukaryotes play key roles in the functioning of marine ecosystems, but we still have a limited knowledge on their ecology and evolution. The MAST-4 lineage is of particular interest, since it is widespread in surface oceans, presents ecotypic differentiation and has defied culturing efforts so far. Single cell genomics (SCG) are promising tools to retrieve genomic information from these uncultured organisms. However, SCG are based on whole genome amplification, which normally introduces amplification biases that limit the amount of genomic data retrieved from a single cell. Here, we increase the recovery of genomic information from two MAST-4 lineages by co-assembling short reads from multiple Single Amplified Genomes (SAGs) belonging to evolutionary closely related cells. We found that complementary genomic information is retrieved from different SAGs, generating co-assembly that features >74% of genome recovery, against about 20% when assembled individually. Even though this approach is not aimed at generating high-quality draft genomes, it allows accessing to the genomic information of microbes that would otherwise remain unreachable. Since most of the picoeukaryotes still remain uncultured, our work serves as a proof-of-concept that can be applied to other taxa in order to extract genomic data and address new ecological and evolutionary questions.

  20. A computational functional genomics based self-limiting self-concentration mechanism of cell specialization as a biological role of jumping genes.

    PubMed

    Lötsch, Jörn; Ultsch, Alfred

    2016-01-01

    Specialization is ubiquitous in biological systems and its manifold mechanisms are active research topics. Although clearly adaptive, the way in which specialization of cells is realized remains incompletely understood as it requires the reshaping of a cell's genome to favor particular biological processes in the competition on a cell's functional capacity. Here, a self-specialization mechanism is identified as a possible biological role of jumping genes, in particular LINE-1 retrotransposition. The mechanism is self-limiting and consistent with its evolutionary preservation despite its likely gene-breaking effects. The scenario we studied was the need for a cell to process a longer exposition to an extraordinary situation, for example continuous exposure to the nociceptive input or the intake of addictive drugs. Both situations may evolve toward chronification. The mechanism involves competition within a gene set in which a subset of genes cooperating in particular biological processes. The subset carries a piece of information, consisting of the LINE-1 sequence, about the destruction of their functional competitor genes which are not involved in that process. During gene transcription, an active copy of LINE-1 is co-transcribed. At a certain low probability, a subsequently transcribed and thus actually exposed gene can be rendered nonfunctional by LINE-1 retrotransposition in a relevant gene part. As retrotransposition needs time it is unlikely that LINE-1 retrotranspose into its own carrier gene. This reshapes the cell genome toward self-specializing of those biological processes that are carried out with a high number of LINE-1 containing genes. Self-termination of the mechanism is achieved by allowing LINE-1 to also occasionally jump into the coding region of itself, thus destroying the information about competitor destruction by successively decreasing the number of LINE-1 until the mechanism ceases. Employing a computational functional genomics approach, we

  1. Calibrating genomic and allelic coverage bias in single-cell sequencing

    PubMed Central

    Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher

    2016-01-01

    Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913

  2. Calibrating genomic and allelic coverage bias in single-cell sequencing.

    PubMed

    Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher

    2015-04-16

    Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.

  3. Development of Advanced Technologies for Complete Genomic and Proteomic Characterization of Quantized Human Tumor Cells

    DTIC Science & Technology

    2014-07-01

    biomarker discovery . To complete this aim, we need blood and PBMC cells from both patients and their family members. Our clinical collaborators at...selection and family history of samples submitted to Complete Genomics for WGS analysis. Table 2. CGI quality control template for 12 genomic DNA...from a few kb long to entire chromosomes – based on comparison of the genome coverage signal to a pre-computed “median coverage profile” of many

  4. CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

    PubMed

    Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

    2016-07-15

    CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

  5. From Genomics to Gene Therapy: Induced Pluripotent Stem Cells Meet Genome Editing.

    PubMed

    Hotta, Akitsu; Yamanaka, Shinya

    2015-01-01

    The advent of induced pluripotent stem (iPS) cells has opened up numerous avenues of opportunity for cell therapy, including the initiation in September 2014 of the first human clinical trial to treat dry age-related macular degeneration. In parallel, advances in genome-editing technologies by site-specific nucleases have dramatically improved our ability to edit endogenous genomic sequences at targeted sites of interest. In fact, clinical trials have already begun to implement this technology to control HIV infection. Genome editing in iPS cells is a powerful tool and enables researchers to investigate the intricacies of the human genome in a dish. In the near future, the groundwork laid by such an approach may expand the possibilities of gene therapy for treating congenital disorders. In this review, we summarize the exciting progress being made in the utilization of genomic editing technologies in pluripotent stem cells and discuss remaining challenges toward gene therapy applications.

  6. Genome Sequence of Mycoplasma hyorhinis Isolated from Cell Cultures

    PubMed Central

    Cibulski, Samuel Paulo; Siqueira, Franciele Maboni; Teixeira, Thais Fumaco; Mayer, Fabiana Quoos; Almeida, Luiz Gonzaga

    2016-01-01

    Mycoplasmas are major contaminants of mammalian cell cultures. Here, the complete genome sequence of Mycoplasma hyorhinis recovered from Madin-Darby bovine kidney (MDBK) cells is reported. PMID:27738034

  7. Genome inside genome: NGS based identification and assembly of endophytic Sphingopyxis granuli and Pseudomonas aeruginosa genomes from rice genomic reads.

    PubMed

    Battu, Latha; Reddy, Mettu Madhavi; Goud, Burragoni Sravanthi; Ulaganathan, Kayalvili; Kandasamy, Ulaganathan

    2017-02-10

    The interactions between crop plants and the endophytic bacteria colonizing them are poorly understood and experimental methods were found to be inadequate to meet the complexities associated with the interaction. Moreover, research on endophytic bacteria was focused at host plant species level and not at cultivar level which is essential for understanding the role played by them on the productivity of specific crop genotype. High throughput genomics offers valuable tools for identification, characterization of endophytic bacteria and understand their interaction with host plants. In this paper we report the use of high throughput plant genomic data for identification of endophytic bacteria colonizing rice plants. Using this novel next generation sequencing based computational method Sphingopyxis granuli and Pseudomonas aeruginosa were identified as endophytes colonizing the elite indica rice cultivar RP Bio-226 and their draft genome sequences were assembled.

  8. Aberrant genomic imprinting in rhesus monkey embryonic stem cells.

    PubMed

    Fujimoto, Akihisa; Mitalipov, Shoukhrat M; Kuo, Hung-Chih; Wolf, Don P

    2006-03-01

    Genomic imprinting involves modification of a gene or a chromosomal region that results in the differential expression of parental alleles. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. Additionally, abnormal imprinting occurs in mouse embryonic stem cells (ESCs) and in clonally derived animals. Imprinted gene expression patterns in primate ESCs are largely unknown, despite the clinical potential of the latter in the cell-based treatment of human disease. Because of the possible implications of abnormal gene expression to cell or tissue replacement therapies involving ESCs, we examined allele specific expression of four imprinted genes in the rhesus macaque. Genomic and complementary DNA from embryos and ESC lines containing useful single nucleotide polymorphisms were subjected to polymerase chain reaction-based amplification and sequence analysis. In blastocysts, NDN expression was variable indicating abnormal or incomplete imprinting whereas IGF2 and SNRPN were expressed exclusively from the paternal allele and H19 from the maternal allele as expected. In ESCs, both NDN and SNRPN were expressed from the paternal allele while IGF2 and H19 showed loss of imprinting and biallelic expression. In differentiated ESC progeny, these expression patterns were maintained. The implications of aberrant imprinted gene expression to ESC differentiation in vitro and on ESC-derived cell function in vivo after transplantation are unknown.

  9. UV Decontamination of MDA Reagents for Single Cell Genomics

    SciTech Connect

    Lee, Janey; Tighe, Damon; Sczyrba, Alexander; Malmatrom, Rex; Clingenpeel, Scott; Malfatti, Stephanie; Rinke, Christian; Wang, Zhong; Stepanauskas, Ramunas; Cheng, Jan-Fang; Woyke, Tanja

    2011-03-18

    Single cell genomics, the amplification and sequencing of genomes from single cells, can provide a glimpse into the genetic make-up and thus life style of the vast majority of uncultured microbial cells, making it an immensely powerful and increasingly popular tool. This is accomplished by use of multiple displacement amplification (MDA), which can generate billions of copies of a single bacterial genome producing microgram-range DNA required for shotgun sequencing. Here, we address a key challenge inherent to this approach and propose a solution for the improved recovery of single cell genomes. While DNA-free reagents for the amplification of a single cell genome are a prerequisite for successful single cell sequencing and analysis, DNA contamination has been detected in various reagents, which poses a considerable challenge. Our study demonstrates the effect of UV irradiation in efficient elimination of exogenous contaminant DNA found in MDA reagents, while maintaining Phi29 activity. Consequently, we also find that increased UV exposure to Phi29 does not adversely affect genome coverage of MDA amplified single cells. While additional challenges in single cell genomics remain to be resolved, the proposed methodology is relatively quick and simple and we believe that its application will be of high value for future single cell sequencing projects.

  10. CD8+ T cell recognition of epitopes within the capsid of adeno-associated virus 8-based gene transfer vectors depends on vectors' genome.

    PubMed

    Wu, Te-Lang; Li, Hua; Faust, Susan M; Chi, Emily; Zhou, Shangzhen; Wright, Fraser; High, Katherine A; Ertl, Hildegund C J

    2014-01-01

    Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8+ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8+ T cells. Our results clearly show that depending on the vectors' genome, CD8+ T cells can detect such epitopes on AAV8's capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.

  11. Single cell genome analysis of an uncultured heterotrophic stramenopile

    NASA Astrophysics Data System (ADS)

    Roy, Rajat S.; Price, Dana C.; Schliep, Alexander; Cai, Guohong; Korobeynikov, Anton; Yoon, Hwan Su; Yang, Eun Chan; Bhattacharya, Debashish

    2014-04-01

    A broad swath of eukaryotic microbial biodiversity cannot be cultivated in the lab and is therefore inaccessible to conventional genome-wide comparative methods. One promising approach to study these lineages is single cell genomics (SCG), whereby an individual cell is captured from nature and genome data are produced from the amplified total DNA. Here we tested the efficacy of SCG to generate a draft genome assembly from a single sample, in this case a cell belonging to the broadly distributed MAST-4 uncultured marine stramenopiles. Using de novo gene prediction, we identified 6,996 protein-encoding genes in the MAST-4 genome. This genetic inventory was sufficient to place the cell within the ToL using multigene phylogenetics and provided preliminary insights into the complex evolutionary history of horizontal gene transfer (HGT) in the MAST-4 lineage.

  12. Single cell genome analysis of an uncultured heterotrophic stramenopile

    PubMed Central

    Roy, Rajat S.; Price, Dana C.; Schliep, Alexander; Cai, Guohong; Korobeynikov, Anton; Yoon, Hwan Su; Yang, Eun Chan; Bhattacharya, Debashish

    2014-01-01

    A broad swath of eukaryotic microbial biodiversity cannot be cultivated in the lab and is therefore inaccessible to conventional genome-wide comparative methods. One promising approach to study these lineages is single cell genomics (SCG), whereby an individual cell is captured from nature and genome data are produced from the amplified total DNA. Here we tested the efficacy of SCG to generate a draft genome assembly from a single sample, in this case a cell belonging to the broadly distributed MAST-4 uncultured marine stramenopiles. Using de novo gene prediction, we identified 6,996 protein-encoding genes in the MAST-4 genome. This genetic inventory was sufficient to place the cell within the ToL using multigene phylogenetics and provided preliminary insights into the complex evolutionary history of horizontal gene transfer (HGT) in the MAST-4 lineage. PMID:24759094

  13. Genome annotation in a community college cell biology lab.

    PubMed

    Beagley, C Timothy

    2013-01-01

    The Biology Department at Salt Lake Community College has used the IMG-ACT toolbox to introduce a genome mapping and annotation exercise into the laboratory portion of its Cell Biology course. This project provides students with an authentic inquiry-based learning experience while introducing them to computational biology and contemporary learning skills. Additionally, the project strengthens student understanding of the scientific method and contributes to student learning gains in curricular objectives centered around basic molecular biology, specifically, the Central Dogma. Importantly, inclusion of this project in the laboratory course provides students with a positive learning environment and allows for the use of cooperative learning strategies to increase overall student success.

  14. Integrated Genome-Based Studies of Shewanella Echophysiology

    SciTech Connect

    Margrethe H. Serres

    2012-06-29

    Shewanella oneidensis MR-1 is a motile, facultative {gamma}-Proteobacterium with remarkable respiratory versatility; it can utilize a range of organic and inorganic compounds as terminal electronacceptors for anaerobic metabolism. The ability to effectively reduce nitrate, S0, polyvalent metals andradionuclides has established MR-1 as an important model dissimilatory metal-reducing microorganism for genome-based investigations of biogeochemical transformation of metals and radionuclides that are of concern to the U.S. Department of Energy (DOE) sites nationwide. Metal-reducing bacteria such as Shewanella also have a highly developed capacity for extracellular transfer of respiratory electrons to solid phase Fe and Mn oxides as well as directly to anode surfaces in microbial fuel cells. More broadly, Shewanellae are recognized free-living microorganisms and members of microbial communities involved in the decomposition of organic matter and the cycling of elements in aquatic and sedimentary systems. To function and compete in environments that are subject to spatial and temporal environmental change, Shewanella must be able to sense and respond to such changes and therefore require relatively robust sensing and regulation systems. The overall goal of this project is to apply the tools of genomics, leveraging the availability of genome sequence for 18 additional strains of Shewanella, to better understand the ecophysiology and speciation of respiratory-versatile members of this important genus. To understand these systems we propose to use genome-based approaches to investigate Shewanella as a system of integrated networks; first describing key cellular subsystems - those involved in signal transduction, regulation, and metabolism - then building towards understanding the function of whole cells and, eventually, cells within populations. As a general approach, this project will employ complimentary "top-down" - bioinformatics-based genome functional predictions, high

  15. Distinct p53 genomic binding patterns in normal and cancer-derived human cells

    SciTech Connect

    Botcheva K.; McCorkle S. R.; McCombie W. R.; Dunn J. J.; Anderson C. W.

    2011-12-15

    We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40% were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied, functional p53 binding sites and, to date, not observed by previous p53 genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands, in marked contrast to sites reported in cancer-derived cells. The distinct genomic features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely, the different chromatin landscape in normal, compared with cancer-derived cells, influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIPseq peaks to the recently published IMR90 methylome1 and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells.

  16. Mechanisms of Base Substitution Mutagenesis in Cancer Genomes

    PubMed Central

    Bacolla, Albino; Cooper, David N.; Vasquez, Karen M.

    2014-01-01

    Cancer genome sequence data provide an invaluable resource for inferring the key mechanisms by which mutations arise in cancer cells, favoring their survival, proliferation and invasiveness. Here we examine recent advances in understanding the molecular mechanisms responsible for the predominant type of genetic alteration found in cancer cells, somatic single base substitutions (SBSs). Cytosine methylation, demethylation and deamination, charge transfer reactions in DNA, DNA replication timing, chromatin status and altered DNA proofreading activities are all now known to contribute to the mechanisms leading to base substitution mutagenesis. We review current hypotheses as to the major processes that give rise to SBSs and evaluate their relative relevance in the light of knowledge acquired from cancer genome sequencing projects and the study of base modifications, DNA repair and lesion bypass. Although gene expression data on APOBEC3B enzymes provide support for a role in cancer mutagenesis through U:G mismatch intermediates, the enzyme preference for single-stranded DNA may limit its activity genome-wide. For SBSs at both CG:CG and YC:GR sites, we outline evidence for a prominent role of damage by charge transfer reactions that follow interactions of the DNA with reactive oxygen species (ROS) and other endogenous or exogenous electron-abstracting molecules. PMID:24705290

  17. Recent advances in microbial single cell genomics technology and applications

    NASA Astrophysics Data System (ADS)

    Stepanauskas, R.

    2015-12-01

    Single cell genomics is increasingly utilized as a powerful tool to decipher the metabolic potential, evolutionary histories and in situ interactions of environmental microorganisms. I will present several new developments of this exciting technology, which improve genomic data recovery from individual cells and allow its integration with cell's phenotypic properties. I will also demonstrate how these new technical capabilities help understanding the biology of the "microbial dark matter" inhabiting marine and terrestrial subsurface environments.

  18. A Single-Cell Genome for Thiovulum sp.

    PubMed Central

    Marshall, Ian P. G.; Blainey, Paul C.

    2012-01-01

    We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 μm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes. PMID:23023751

  19. Prognostic value of genomic damage in non-small-cell lung cancer.

    PubMed

    de Juan, C; Iniesta, P; Vega, F J; Peinado, M A; Fernandez, C; Caldés, T; Massa, M J; López, J A; Sánchez, A; Torres, A J; Balibrea, J L; Benito, M

    1998-06-01

    Genomic alterations have been analysed in 65 non-small-cell lung cancer (NSCLC) tissue samples by using the arbitrarily primed polymerase chain reaction (AP-PCR), which is a PCR-based genomic fingerprinting. We have shown that AP-PCR may be applied as a useful and feasible practical method for detection of the genomic alterations that accompany malignancy in NSCLC. Genomic changes detected by us consisted of: allelic losses or gains in anonymous DNA sequences, homozygously deleted DNA sequences and polymorphic DNA sequences. According to these genomic changes, lung tumours evaluated in the present study have been scored into three groups: low, moderate and high genomic damage tumours. The aim of this study was to investigate the effect of genomic damage on patient survival. Survival analysis was carried out in 51 NSCLC patients. Our results revealed that high genomic damage patients showed a poorer prognosis than those with low or moderate genomic damage (P = 0.038). Multivariate Cox regression analysis showed that patients with higher genomic alterations displayed an adjusted-by-stage risk ratio 4.26 times higher than the remaining patients (95% CI = 1.03-17.54). We can conclude that genomic damage has an independent prognostic value of poor clinical evolution in NSCLC.

  20. Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

    PubMed

    Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

    2010-10-01

    Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens.

  1. GenColors-based comparative genome databases for small eukaryotic genomes.

    PubMed

    Felder, Marius; Romualdi, Alessandro; Petzold, Andreas; Platzer, Matthias; Sühnel, Jürgen; Glöckner, Gernot

    2013-01-01

    Many sequence data repositories can give a quick and easily accessible overview on genomes and their annotations. Less widespread is the possibility to compare related genomes with each other in a common database environment. We have previously described the GenColors database system (http://gencolors.fli-leibniz.de) and its applications to a number of bacterial genomes such as Borrelia, Legionella, Leptospira and Treponema. This system has an emphasis on genome comparison. It combines data from related genomes and provides the user with an extensive set of visualization and analysis tools. Eukaryote genomes are normally larger than prokaryote genomes and thus pose additional challenges for such a system. We have, therefore, adapted GenColors to also handle larger datasets of small eukaryotic genomes and to display eukaryotic gene structures. Further recent developments include whole genome views, genome list options and, for bacterial genome browsers, the display of horizontal gene transfer predictions. Two new GenColors-based databases for two fungal species (http://fgb.fli-leibniz.de) and for four social amoebas (http://sacgb.fli-leibniz.de) were set up. Both new resources open up a single entry point for related genomes for the amoebozoa and fungal research communities and other interested users. Comparative genomics approaches are greatly facilitated by these resources.

  2. GenColors-based comparative genome databases for small eukaryotic genomes

    PubMed Central

    Felder, Marius; Romualdi, Alessandro; Petzold, Andreas; Platzer, Matthias; Sühnel, Jürgen; Glöckner, Gernot

    2013-01-01

    Many sequence data repositories can give a quick and easily accessible overview on genomes and their annotations. Less widespread is the possibility to compare related genomes with each other in a common database environment. We have previously described the GenColors database system (http://gencolors.fli-leibniz.de) and its applications to a number of bacterial genomes such as Borrelia, Legionella, Leptospira and Treponema. This system has an emphasis on genome comparison. It combines data from related genomes and provides the user with an extensive set of visualization and analysis tools. Eukaryote genomes are normally larger than prokaryote genomes and thus pose additional challenges for such a system. We have, therefore, adapted GenColors to also handle larger datasets of small eukaryotic genomes and to display eukaryotic gene structures. Further recent developments include whole genome views, genome list options and, for bacterial genome browsers, the display of horizontal gene transfer predictions. Two new GenColors-based databases for two fungal species (http://fgb.fli-leibniz.de) and for four social amoebas (http://sacgb.fli-leibniz.de) were set up. Both new resources open up a single entry point for related genomes for the amoebozoa and fungal research communities and other interested users. Comparative genomics approaches are greatly facilitated by these resources. PMID:23193285

  3. Ten years of bacterial genome sequencing: comparative-genomics-based discoveries.

    PubMed

    Binnewies, Tim T; Motro, Yair; Hallin, Peter F; Lund, Ole; Dunn, David; La, Tom; Hampson, David J; Bellgard, Matthew; Wassenaar, Trudy M; Ussery, David W

    2006-07-01

    It has been more than 10 years since the first bacterial genome sequence was published. Hundreds of bacterial genome sequences are now available for comparative genomics, and searching a given protein against more than a thousand genomes will soon be possible. The subject of this review will address a relatively straightforward question: "What have we learned from this vast amount of new genomic data?" Perhaps one of the most important lessons has been that genetic diversity, at the level of large-scale variation amongst even genomes of the same species, is far greater than was thought. The classical textbook view of evolution relying on the relatively slow accumulation of mutational events at the level of individual bases scattered throughout the genome has changed. One of the most obvious conclusions from examining the sequences from several hundred bacterial genomes is the enormous amount of diversity--even in different genomes from the same bacterial species. This diversity is generated by a variety of mechanisms, including mobile genetic elements and bacteriophages. An examination of the 20 Escherichia coli genomes sequenced so far dramatically illustrates this, with the genome size ranging from 4.6 to 5.5 Mbp; much of the variation appears to be of phage origin. This review also addresses mobile genetic elements, including pathogenicity islands and the structure of transposable elements. There are at least 20 different methods available to compare bacterial genomes. Metagenomics offers the chance to study genomic sequences found in ecosystems, including genomes of species that are difficult to culture. It has become clear that a genome sequence represents more than just a collection of gene sequences for an organism and that information concerning the environment and growth conditions for the organism are important for interpretation of the genomic data. The newly proposed Minimal Information about a Genome Sequence standard has been developed to obtain this

  4. Array-comparative genomic hybridization profiling of immunohistochemical subgroups of diffuse large B-cell lymphoma shows distinct genomic alterations

    PubMed Central

    Guo, Ying; Takeuchi, Ichiro; Karnan, Sivasundaram; Miyata, Tomoko; Ohshima, Koichi; Seto, Masao

    2014-01-01

    Diffuse large B-cell lymphoma (DLBCL) displays striking heterogeneity at the clinical, genetic and molecular levels. Subtypes include germinal center B-cell-like (GCB) DLBCL and activated B-cell-like (ABC) DLBCL, according to microarray analysis, and germinal center type or non-germinal center type by immunohistochemistry. Although some reports have described genomic aberrations based upon microarray classification system, genomic aberrations based upon immunohistochemical classifications have rarely been reported. The present study aimed to ascertain the relationship between genomic aberrations and subtypes identified by immunohistochemistry, and to study the pathogenetic character of Chinese DLBCL. We conducted immunohistochemistry using antibodies against CD10, BCL6 and MUM1 in 59 samples of DLBCL from Chinese patients, and then performed microarray-based comparative genomic hybridization for each case. Characteristic genomic differences were found between GCB and non-GCB DLBCL from the array data. The GCB type was characterized by more gains at 7q (7q22.1, P < 0.05) and losses at 16q (P ≤ 0.05), while the non-GCB type was characterized by gains at 11q24.3 and 3q13.2 (P < 0.05). We found completely different mutations in BCL6+ and BCL6− non-GCB type DLBCL, whereby the BCL6− group had a higher number of gains at 1q and a loss at 14q32.13 (P ≤ 0.005), while the BCL6+ group showed a higher number of gains at 14q23.1 (P = 0.15) and losses at 6q (P = 0.07). The BCL6− group had a higher frequency of genomic imbalances compared to the BCL6+ group. In conclusion, the BCL6+ and BCL6− non-GCB type of DLBCL appear to have different mechanisms of pathogenesis. PMID:24843885

  5. Genome-wide analysis of histone methylation reveals chromatin state-based complex regulation of differential gene transcription and function of CD8 memory T cells

    PubMed Central

    Araki, Yasuto; Wang, Zhibin; Zang, Chongzhi; Wood, William H.; Schones, Dustin; Cui, Kairong; Roh, Tae-Young; Lhotsky, Brad; Wersto, Robert P.; Peng, Weiqun; Becker, Kevin G.; Zhao, Keji; Weng, Nan-ping

    2009-01-01

    Summary Memory lymphocytes are characterized by their ability to exhibit a rapid response to the recall antigen, in which differential transcription plays a significant role, yet the underlying mechanism is not understood. We report here a genome-wide analysis of histone methylation on two histone H3 lysine residues (H3K4me3 and H3K27me3) and gene expression profiles in naïve and memory CD8 T cells. We found that a general correlation exists between the levels of gene expression and the levels of H3K4me3 (positive correlation) and H3K27me3 (negative correlation) across the gene body. These correlations display four distinct modes: repressive, active, poised, and bivalent, reflecting different functions of these genes. Furthermore, a permissive chromatin state of each gene is established by a combination of different histone modifications. Our findings reveal a complex regulation by histone methylation in differential gene expression and suggest that histone methylation may be responsible for memory CD8 T cell function. PMID:19523850

  6. Weighted gene co-expression network analysis in identification of metastasis-related genes of lung squamous cell carcinoma based on the Cancer Genome Atlas database

    PubMed Central

    Tian, Feng; Zhao, Jinlong; Kang, Zhenxing

    2017-01-01

    Background Lung squamous cell carcinoma (lung SCC) is a common type of malignancy. Its pathogenesis mechanism of tumor development is unclear. The aim of this study was to identify key genes for diagnosis biomarkers in lung SCC metastasis. Methods We searched and downloaded mRNA expression data and clinical data from The Cancer Genome Atlas (TCGA) database to identify differences in mRNA expression of primary tumor tissues from lung SCC with and without metastasis. Gene co-expression network analysis, protein-protein interaction (PPI) network, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and quantitative real-time polymerase chain reactions (qRT-PCR) were used to explore the biological functions of the identified dysregulated genes. Results Four hundred and eighty-two differentially expressed genes (DEGs) were identified between lung SCC with and without metastasis. Nineteen modules were identified in lung SCC through weighted gene co-expression network analysis (WGCNA). Twenty-three DEGs and 26 DEGs were significantly enriched in the respective pink and black module. KEGG pathway analysis displayed that 26 DEGs in the black module were significantly enriched in bile secretion pathway. Forty-nine DEGs in the two gene co-expression module were used to construct PPI network. CFTR in the black module was the hub protein, had the connectivity with 182 genes. The results of qRT-PCR displayed that FIGF, SFTPD, DYNLRB2 were significantly down-regulated in the tumor samples of lung SCC with metastasis and CFTR, SCGB3A2, SSTR1, SCTR, ROPN1L had the down-regulation tendency in lung SCC with metastasis compared to lung SCC without metastasis. Conclusions The dysregulated genes including CFTR, SCTR and FIGF might be involved in the pathology of lung SCC metastasis and could be used as potential diagnosis biomarkers or therapeutic targets for lung SCC. PMID:28203405

  7. Array-Based Comparative Genomic Hybridization Analysis Reveals Chromosomal Copy Number Aberrations Associated with Clinical Outcome in Canine Diffuse Large B-Cell Lymphoma

    PubMed Central

    Bresolin, Silvia; Marconato, Laura; Comazzi, Stefano; Te Kronnie, Geertruy; Aresu, Luca

    2014-01-01

    Canine Diffuse Large B-cell Lymphoma (cDLBCL) is an aggressive cancer with variable clinical response. Despite recent attempts by gene expression profiling to identify the dog as a potential animal model for human DLBCL, this tumor remains biologically heterogeneous with no prognostic biomarkers to predict prognosis. The aim of this work was to identify copy number aberrations (CNAs) by high-resolution array comparative genomic hybridization (aCGH) in 12 dogs with newly diagnosed DLBCL. In a subset of these dogs, the genetic profiles at the end of therapy and at relapse were also assessed. In primary DLBCLs, 90 different genomic imbalances were counted, consisting of 46 gains and 44 losses. Two gains in chr13 were significantly correlated with clinical stage. In addition, specific regions of gains and losses were significantly associated to duration of remission. In primary DLBCLs, individual variability was found, however 14 recurrent CNAs (>30%) were identified. Losses involving IGK, IGL and IGH were always found, and gains along the length of chr13 and chr31 were often observed (>41%). In these segments, MYC, LDHB, HSF1, KIT and PDGFRα are annotated. At the end of therapy, dogs in remission showed four new CNAs, whereas three new CNAs were observed in dogs at relapse compared with the previous profiles. One ex novo CNA, involving TCR, was present in dogs in remission after therapy, possibly induced by the autologous vaccine. Overall, aCGH identified small CNAs associated with outcome, which, along with future expression studies, may reveal target genes relevant to cDLBCL. PMID:25372838

  8. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

    SciTech Connect

    Gorman, C.M.; Moffat, L.F.; Howard, B.H.

    1982-09-01

    The authors constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV 40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. They also constructed a recombinant, pSVO-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

  9. Genome size and DNA base composition of geophytes: the mirror of phenology and ecology?

    PubMed Central

    Veselý, Pavel; Bureš, Petr; Šmarda, Petr; Pavlíček, Tomáš

    2012-01-01

    Background and Aims Genome size is known to affect various plant traits such as stomatal size, seed mass, and flower or shoot phenology. However, these associations are not well understood for species with very large genomes, which are laregly represented by geophytic plants. No detailed associations are known between DNA base composition and genome size or species ecology. Methods Genome sizes and GC contents were measured in 219 geophytes together with tentative morpho-anatomical and ecological traits. Key Results Increased genome size was associated with earliness of flowering and tendency to grow in humid conditions, and there was a positive correlation between an increase in stomatal size in species with extremely large genomes. Seed mass of geophytes was closely related to their ecology, but not to genomic parameters. Genomic DNA GC content showed a unimodal relationship with genome size but no relationship with species ecology. Conclusions Evolution of genome size in geophytes is closely related to their ecology and phenology and is also associated with remarkable changes in DNA base composition. Although geophytism together with producing larger cells appears to be an advantageous strategy for fast development of an organism in seasonal habitats, the drought sensitivity of large stomata may restrict the occurrence of geophytes with very large genomes to regions not subject to water stress. PMID:22021815

  10. Genomics and public health: development of Web-based training tools for increasing genomic awareness.

    PubMed

    Bodzin, Jennifer; Kardia, Sharon L R; Goldenberg, Aaron; Raup, Sarah F; Bach, Janice V; Citrin, Toby

    2005-04-01

    In 2001, the Centers for Disease Control and Prevention funded three Centers for Genomics and Public Health to develop training tools for increasing genomic awareness. Over the past three years, the centers, working together with the Centers for Disease Control and Prevention's Office of Genomics and Disease Prevention, have developed tools to increase awareness of the impact genomics will have on public health practice, to provide a foundation for understanding basic genomic advances, and to translate the relevance of that information to public health practitioners' own work. These training tools serve to communicate genomic advances and their potential for integration into public heath practice. This paper highlights two of these training tools: 1) Genomics for Public Health Practitioners: The Practical Application of Genomics in Public Health Practice, a Web-based introduction to genomics, and 2) Six Weeks to Genomic Awareness, an in-depth training module on public health genomics. This paper focuses on the processes and collaborative efforts by which these live presentations were developed and delivered as Web-based training sessions.

  11. CyanoBase: the cyanobacteria genome database update 2010.

    PubMed

    Nakao, Mitsuteru; Okamoto, Shinobu; Kohara, Mitsuyo; Fujishiro, Tsunakazu; Fujisawa, Takatomo; Sato, Shusei; Tabata, Satoshi; Kaneko, Takakazu; Nakamura, Yasukazu

    2010-01-01

    CyanoBase (http://genome.kazusa.or.jp/cyanobase) is the genome database for cyanobacteria, which are model organisms for photosynthesis. The database houses cyanobacteria species information, complete genome sequences, genome-scale experiment data, gene information, gene annotations and mutant information. In this version, we updated these datasets and improved the navigation and the visual display of the data views. In addition, a web service API now enables users to retrieve the data in various formats with other tools, seamlessly.

  12. The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops

    PubMed Central

    de Belle, Ian; Cai, Shutao; Kohwi-Shigematsu, Terumi

    1998-01-01

    Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1–1.1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island–containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear “halos” with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type–specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell–specific gene regulation. PMID:9548713

  13. Differentiation and Genomic Instability in a Human Mammary Cell Model

    NASA Technical Reports Server (NTRS)

    Richmond, R.; Kale, R.; Pettengill, O.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Harvest of prophylactic mastectomy specimens from an obligate heterozygote for ataxia-telangiectasia provided autologous fibroblasts as well epithelial cells (HMEC). The routine availability of these autologous cells has provided an opportunity to study cell-cell interactions in coculture and monoculture, and in 3-dimensional cultures grown in the NASA rotating bioreactor. HMEC and stromal fibroblasts grown in 2-dimensional monoculture were both observed to produce extracellular matrix. Similar matrix was encountered in 3-dimensional cultures containing HMEC. Metaphases were analyzed. For stromal fibroblasts, genomic aberrations were found in 18% of metaphase spreads. For HMEC, aberrations were greater such that a majority were found to be abnormal. The level of genomic instability determined for these noncancerous cells in 2-dimensional monoculture should be useful for generating a human cell model that can correlate the effects of differentiation in 3-dimensional coculture on the level of genomic instability.

  14. Genome damage in induced pluripotent stem cells: assessing the mechanisms and their consequences.

    PubMed

    Hussein, Samer M I; Elbaz, Judith; Nagy, Andras A

    2013-03-01

    In 2006, Shinya Yamanaka and colleagues discovered how to reprogram terminally differentiated somatic cells to a pluripotent stem cell state. The resulting induced pluripotent stem cells (iPSCs) made a paradigm shift in the field, further nailing down the disproval of the long-held dogma that differentiation is unidirectional. The prospect of using iPSCs for patient-specific cell-based therapies has been enticing. This promise, however, has been questioned in the last two years as several studies demonstrated intrinsic epigenetic and genomic anomalies in these cells. Here, we not only review the recent critical studies addressing the genome integrity during the reprogramming process, but speculate about the underlying mechanisms that could create de novo genome damage in iPSCs. Finally, we discuss how much an elevated mutation load really matters considering the safety of future therapies with cells heavily cultured in vitro.

  15. FISH 'N' Chips : a single cell genomic analyzer for the human microbiome.

    SciTech Connect

    Light, Yooli Kim; Perroud, Thomas D.; Hugenholtz, Philip; Meagher, Robert J.; Singh, Anup K.; Malamud, Daniel; Saxena, Deepak; Liu, Peng

    2010-09-01

    Uncultivable microorganisms likely play significant roles in the ecology within the human body, with subtle but important implications for human health. Focusing on the oral microbiome, we are developing a processor for targeted isolation of individual microbial cells, facilitating whole-genome analysis without the need for isolation of pure cultures. The processor consists of three microfluidic modules: identification based on 16S rRNA fluorescence in situ hybridization (FISH), fluorescence-based sorting, and encapsulation of individual selected cells into small droplets for whole genome amplification. We present here a technique for performing microscale FISH and flow cytometry, as a prelude to single cell sorting.

  16. Low-dose carbon-based nanoparticle-induced effects in A549 lung cells determined by biospectroscopy are associated with increases in genomic methylation

    NASA Astrophysics Data System (ADS)

    Li, Junyi; Tian, Meiping; Cui, Li; Dwyer, John; Fullwood, Nigel J.; Shen, Heqing; Martin, Francis L.

    2016-02-01

    Nanotechnology has introduced many manufactured carbon-based nanoparticles (CNPs) into our environment, generating a debate into their risks and benefits. Numerous nanotoxicology investigations have been carried, and nanoparticle-induced toxic effects have been reported. However, there remain gaps in our knowledge, primarily regarding mechanism. Herein, we assessed the global alterations induced by CNPs in A549 lung cells using biospectroscopy techniques, including attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and surface-enhanced Raman spectroscopy (SERS). A549 cells were treated with fullerene (C60), long or short multi-walled carbon nanotubes, or single-walled carbon nanotubes at concentrations of 0.1 mg/L, 0.01 mg/L and 0.001 mg/L. Exposed cells were then analysed by ATR-FTIR spectroscopy and SERS. Spectra were pre-processed via computational analysis, and information on biochemical alterations in exposed cells were identified. Additionally, global DNA methylation levels in cells exposed to CNPs at 0.1 mg/L were determined using HPLC-MS and genetic regulators (for DNA methylation) were checked by quantitative real-time RT-PCR. It was found that CNPs exert marked effects in A549 cells and also contribute to increases in global DNA methylation. For the first time, this study highlights that real-world levels of nanoparticles can alter the methylome of exposed cells; this could have enormous implications for their regulatory assessment.

  17. Low-dose carbon-based nanoparticle-induced effects in A549 lung cells determined by biospectroscopy are associated with increases in genomic methylation

    PubMed Central

    Li, Junyi; Tian, Meiping; Cui, Li; Dwyer, John; Fullwood, Nigel J.; Shen, Heqing; Martin, Francis L.

    2016-01-01

    Nanotechnology has introduced many manufactured carbon-based nanoparticles (CNPs) into our environment, generating a debate into their risks and benefits. Numerous nanotoxicology investigations have been carried, and nanoparticle-induced toxic effects have been reported. However, there remain gaps in our knowledge, primarily regarding mechanism. Herein, we assessed the global alterations induced by CNPs in A549 lung cells using biospectroscopy techniques, including attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and surface-enhanced Raman spectroscopy (SERS). A549 cells were treated with fullerene (C60), long or short multi-walled carbon nanotubes, or single-walled carbon nanotubes at concentrations of 0.1 mg/L, 0.01 mg/L and 0.001 mg/L. Exposed cells were then analysed by ATR-FTIR spectroscopy and SERS. Spectra were pre-processed via computational analysis, and information on biochemical alterations in exposed cells were identified. Additionally, global DNA methylation levels in cells exposed to CNPs at 0.1 mg/L were determined using HPLC-MS and genetic regulators (for DNA methylation) were checked by quantitative real-time RT-PCR. It was found that CNPs exert marked effects in A549 cells and also contribute to increases in global DNA methylation. For the first time, this study highlights that real-world levels of nanoparticles can alter the methylome of exposed cells; this could have enormous implications for their regulatory assessment. PMID:26831369

  18. Nuclear envelope and genome interactions in cell fate

    PubMed Central

    Talamas, Jessica A.; Capelson, Maya

    2015-01-01

    The eukaryotic cell nucleus houses an organism’s genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate. PMID:25852741

  19. Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5'-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi.

    PubMed

    Ishikawa, Ken; Watanabe, Miki; Kuroita, Toshihiro; Uchiyama, Ikuo; Bujnicki, Janusz M; Kawakami, Bunsei; Tanokura, Masaru; Kobayashi, Ichizo

    2005-07-21

    To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction-modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5'-GTAC and leave a 3'-TA overhang (5'-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90 degrees C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.

  20. Genomic Sequencing of Single Microbial Cells from Environmental Samples

    SciTech Connect

    Ishoey, Thomas; Woyke, Tanja; Stepanauskas, Ramunas; Novotny, Mark; Lasken, Roger S.

    2008-02-01

    Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification, Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.

  1. Whole-genome molecular haplotyping of single cells.

    PubMed

    Fan, H Christina; Wang, Jianbin; Potanina, Anastasia; Quake, Stephen R

    2011-01-01

    Conventional experimental methods of studying the human genome are limited by the inability to independently study the combination of alleles, or haplotype, on each of the homologous copies of the chromosomes. We developed a microfluidic device capable of separating and amplifying homologous copies of each chromosome from a single human metaphase cell. Single-nucleotide polymorphism (SNP) array analysis of amplified DNA enabled us to achieve completely deterministic, whole-genome, personal haplotypes of four individuals, including a HapMap trio with European ancestry (CEU) and an unrelated European individual. The phases of alleles were determined at ∼99.8% accuracy for up to ∼96% of all assayed SNPs. We demonstrate several practical applications, including direct observation of recombination events in a family trio, deterministic phasing of deletions in individuals and direct measurement of the human leukocyte antigen haplotypes of an individual. Our approach has potential applications in personal genomics, single-cell genomics and statistical genetics.

  2. Concise review: Human cell engineering: cellular reprogramming and genome editing.

    PubMed

    Mali, Prashant; Cheng, Linzhao

    2012-01-01

    Cell engineering is defined here as the collective ability to both reset and edit the genome of a mammalian cell. Until recently, this had been extremely challenging to achieve as nontransformed human cells are significantly refractory to both these processes. The recent success in reprogramming somatic cells into induced pluripotent stem cells that are self-renewable in culture, coupled with our increasing ability to effect precise and predesigned genomic editing, now readily permits cellular changes at both the genetic and epigenetic levels. These dual capabilities also make possible the generation of genetically matched, disease-free stem cells from patients for regenerative medicine. The objective of this review is to summarize the key enabling developments on these two rapidly evolving research fronts in human cell engineering, highlight unresolved issues, and outline potential future research directions.

  3. Viruses roll the dice: the stochastic behavior of viral genome molecules accelerates viral adaptation at the cell and tissue levels.

    PubMed

    Miyashita, Shuhei; Ishibashi, Kazuhiro; Kishino, Hirohisa; Ishikawa, Masayuki

    2015-03-01

    , which are based on the stochastic behavior of the viral genome molecules in each cell.

  4. Ontology-Based Search of Genomic Metadata.

    PubMed

    Fernandez, Javier D; Lenzerini, Maurizio; Masseroli, Marco; Venco, Francesco; Ceri, Stefano

    2016-01-01

    The Encyclopedia of DNA Elements (ENCODE) is a huge and still expanding public repository of more than 4,000 experiments and 25,000 data files, assembled by a large international consortium since 2007; unknown biological knowledge can be extracted from these huge and largely unexplored data, leading to data-driven genomic, transcriptomic, and epigenomic discoveries. Yet, search of relevant datasets for knowledge discovery is limitedly supported: metadata describing ENCODE datasets are quite simple and incomplete, and not described by a coherent underlying ontology. Here, we show how to overcome this limitation, by adopting an ENCODE metadata searching approach which uses high-quality ontological knowledge and state-of-the-art indexing technologies. Specifically, we developed S.O.S. GeM (http://www.bioinformatics.deib.polimi.it/SOSGeM/), a system supporting effective semantic search and retrieval of ENCODE datasets. First, we constructed a Semantic Knowledge Base by starting with concepts extracted from ENCODE metadata, matched to and expanded on biomedical ontologies integrated in the well-established Unified Medical Language System. We prove that this inference method is sound and complete. Then, we leveraged the Semantic Knowledge Base to semantically search ENCODE data from arbitrary biologists' queries. This allows correctly finding more datasets than those extracted by a purely syntactic search, as supported by the other available systems. We empirically show the relevance of found datasets to the biologists' queries.

  5. Hellbender genome sequences shed light on genomic expansion at the base of crown salamanders.

    PubMed

    Sun, Cheng; Mueller, Rachel Lockridge

    2014-07-01

    Among animals, genome sizes range from 20 Mb to 130 Gb, with 380-fold variation across vertebrates. Most of the largest vertebrate genomes are found in salamanders, an amphibian clade of 660 species. Thus, salamanders are an important system for studying causes and consequences of genomic gigantism. Previously, we showed that plethodontid salamander genomes accumulate higher levels of long terminal repeat (LTR) retrotransposons than do other vertebrates, although the evolutionary origins of such sequences remained unexplored. We also showed that some salamanders in the family Plethodontidae have relatively slow rates of DNA loss through small insertions and deletions. Here, we present new data from Cryptobranchus alleganiensis, the hellbender. Cryptobranchus and Plethodontidae span the basal phylogenetic split within salamanders; thus, analyses incorporating these taxa can shed light on the genome of the ancestral crown salamander lineage, which underwent expansion. We show that high levels of LTR retrotransposons likely characterize all crown salamanders, suggesting that disproportionate expansion of this transposable element (TE) class contributed to genomic expansion. Phylogenetic and age distribution analyses of salamander LTR retrotransposons indicate that salamanders' high TE levels reflect persistence and diversification of ancestral TEs rather than horizontal transfer events. Finally, we show that relatively slow DNA loss rates through small indels likely characterize all crown salamanders, suggesting that a decreased DNA loss rate contributed to genomic expansion at the clade's base. Our identification of shared genomic features across phylogenetically distant salamanders is a first step toward identifying the evolutionary processes underlying accumulation and persistence of high levels of repetitive sequence in salamander genomes.

  6. Comprehensive genomic characterization of squamous cell lung cancers.

    PubMed

    2012-09-27

    Lung squamous cell carcinoma is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in squamous cell lung cancers have not been comprehensively characterized, and no molecularly targeted agents have been specifically developed for its treatment. As part of The Cancer Genome Atlas, here we profile 178 lung squamous cell carcinomas to provide a comprehensive landscape of genomic and epigenomic alterations. We show that the tumour type is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumour. We find statistically recurrent mutations in 11 genes, including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations are seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2 and KEAP1 in 34%, squamous differentiation genes in 44%, phosphatidylinositol-3-OH kinase pathway genes in 47%, and CDKN2A and RB1 in 72% of tumours. We identified a potential therapeutic target in most tumours, offering new avenues of investigation for the treatment of squamous cell lung cancers.

  7. Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

    NASA Astrophysics Data System (ADS)

    Hei, T. K.; Piao, C. Q.; Wu, L. J.; Willey, J. C.; Hall, E. J.

    1998-11-01

    Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon 56Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to 56Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.

  8. Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis

    PubMed Central

    Utturkar, Sagar M.; Cude, W. Nathan; Robeson, Michael S.; Yang, Zamin K.; Klingeman, Dawn M.; Land, Miriam L.; Allman, Steve L.; Lu, Tse-Yuan S.; Brown, Steven D.; Schadt, Christopher W.; Podar, Mircea; Doktycz, Mitchel J.

    2016-01-01

    ABSTRACT Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. Here, we describe a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria. IMPORTANCE Plant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses

  9. Genome rearrangement affects RNA virus adaptability on prostate cancer cells.

    PubMed

    Pesko, Kendra; Voigt, Emily A; Swick, Adam; Morley, Valerie J; Timm, Collin; Yin, John; Turner, Paul E

    2015-01-01

    Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3' UTR - core protein genes - envelope protein genes - RNA-dependent RNA-polymerase gene - 5' UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5' end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene

  10. StellaBase: the Nematostella vectensis Genomics Database.

    PubMed

    Sullivan, James C; Ryan, Joseph F; Watson, James A; Webb, Jeramy; Mullikin, James C; Rokhsar, Daniel; Finnerty, John R

    2006-01-01

    StellaBase, the Nematostella vectensis Genomics Database, is a web-based resource that will facilitate desktop and bench-top studies of the starlet sea anemone. Nematostella is an emerging model organism that has already proven useful for addressing fundamental questions in developmental evolution and evolutionary genomics. StellaBase allows users to query the assembled Nematostella genome, a confirmed gene library, and a predicted genome using both keyword and homology based search functions. Data provided by these searches will elucidate gene family evolution in early animals. Unique research tools, including a Nematostella genetic stock library, a primer library, a literature repository and a gene expression library will provide support to the burgeoning Nematostella research community. The development of StellaBase accompanies significant upgrades to CnidBase, the Cnidarian Evolutionary Genomics Database. With the completion of the first sequenced cnidarian genome, genome comparison tools have been added to CnidBase. In addition, StellaBase provides a framework for the integration of additional species-specific databases into CnidBase. StellaBase is available at http://www.stellabase.org.

  11. Genome sequencing of normal cells reveals developmental lineages and mutational processes

    PubMed Central

    Behjati, Sam; Wedge, David C; Tamuri, Asif U; Martincorena, Inigo; Petljak, Mia; Alexandrov, Ludmil B; Gundem, Gunes; Tarpey, Patrick S; Roerink, Sophie; Blokker, Joyce; Maddison, Mark; Mudie, Laura; Robinson, Ben; Nik-Zainal, Serena; Campbell, Peter; Goldman, Nick; van de Wetering, Marc; Cuppen, Edwin; Clevers, Hans; Stratton, Michael R

    2014-01-01

    The somatic mutations present in the genome of a cell have been accumulated over the lifetime of a multicellular organism. These mutations can provide insights into the developmental lineage tree1, the number of divisions each cell has undergone and the mutational processes that have been operative2. Here, we conducted whole genome sequencing of clonal lines3 derived from multiple tissues of healthy mice. Using somatic base substitutions, we reconstructed the early cell divisions of each animal demonstrating the contributions of embryonic cells to adult tissues. Differences were observed between tissues in the numbers and types of mutations accumulated by each cell, which likely reflect differences in the number of cell divisions they have undergone and varying contributions of different mutational processes. If somatic mutation rates are similar to those in mice, the results indicate that precise insights into development and mutagenesis of normal human cells will be possible. PMID:25043003

  12. Genome Informed Trait-Based Models

    NASA Astrophysics Data System (ADS)

    Karaoz, U.; Cheng, Y.; Bouskill, N.; Tang, J.; Beller, H. R.; Brodie, E.; Riley, W. J.

    2013-12-01

    Trait-based approaches are powerful tools for representing microbial communities across both spatial and temporal scales within ecosystem models. Trait-based models (TBMs) represent the diversity of microbial taxa as stochastic assemblages with a distribution of traits constrained by trade-offs between these traits. Such representation with its built-in stochasticity allows the elucidation of the interactions between the microbes and their environment by reducing the complexity of microbial community diversity into a limited number of functional ';guilds' and letting them emerge across spatio-temporal scales. From the biogeochemical/ecosystem modeling perspective, the emergent properties of the microbial community could be directly translated into predictions of biogeochemical reaction rates and microbial biomass. The accuracy of TBMs depends on the identification of key traits of the microbial community members and on the parameterization of these traits. Current approaches to inform TBM parameterization are empirical (i.e., based on literature surveys). Advances in omic technologies (such as genomics, metagenomics, metatranscriptomics, and metaproteomics) pave the way to better-initialize models that can be constrained in a generic or site-specific fashion. Here we describe the coupling of metagenomic data to the development of a TBM representing the dynamics of metabolic guilds from an organic carbon stimulated groundwater microbial community. Illumina paired-end metagenomic data were collected from the community as it transitioned successively through electron-accepting conditions (nitrate-, sulfate-, and Fe(III)-reducing), and used to inform estimates of growth rates and the distribution of metabolic pathways (i.e., aerobic and anaerobic oxidation, fermentation) across a spatially resolved TBM. We use this model to evaluate the emergence of different metabolisms and predict rates of biogeochemical processes over time. We compare our results to observational

  13. A Platform for Designing Genome-Based Personalized Immunotherapy or Vaccine against Cancer

    PubMed Central

    Gupta, Sudheer; Chaudhary, Kumardeep; Dhanda, Sandeep Kumar; Kumar, Rahul; Kumar, Shailesh; Sehgal, Manika; Nagpal, Gandharva

    2016-01-01

    Due to advancement in sequencing technology, genomes of thousands of cancer tissues or cell-lines have been sequenced. Identification of cancer-specific epitopes or neoepitopes from cancer genomes is one of the major challenges in the field of immunotherapy or vaccine development. This paper describes a platform Cancertope, developed for designing genome-based immunotherapy or vaccine against a cancer cell. Broadly, the integrated resources on this platform are apportioned into three precise sections. First section explains a cancer-specific database of neoepitopes generated from genome of 905 cancer cell lines. This database harbors wide range of epitopes (e.g., B-cell, CD8+ T-cell, HLA class I, HLA class II) against 60 cancer-specific vaccine antigens. Second section describes a partially personalized module developed for predicting potential neoepitopes against a user-specific cancer genome. Finally, we describe a fully personalized module developed for identification of neoepitopes from genomes of cancerous and healthy cells of a cancer-patient. In order to assist the scientific community, wide range of tools are incorporated in this platform that includes screening of epitopes against human reference proteome (http://www.imtech.res.in/raghava/cancertope/). PMID:27832200

  14. Recent advances in developing molecular tools for targeted genome engineering of mammalian cells.

    PubMed

    Lim, Kwang-il

    2015-01-01

    Various biological molecules naturally existing in diversified species including fungi, bacteria, and bacteriophage have functionalities for DNA binding and processing. The biological molecules have been recently actively engineered for use in customized genome editing of mammalian cells as the molecule-encoding DNA sequence information and the underlying mechanisms how the molecules work are unveiled. Excitingly, multiple novel methods based on the newly constructed artificial molecular tools have enabled modifications of specific endogenous genetic elements in the genome context at efficiencies that are much higher than that of the conventional homologous recombination based methods. This minireview introduces the most recently spotlighted molecular genome engineering tools with their key features and ongoing modifications for better performance. Such ongoing efforts have mainly focused on the removal of the inherent DNA sequence recognition rigidity from the original molecular platforms, the addition of newly tailored targeting functions into the engineered molecules, and the enhancement of their targeting specificity. Effective targeted genome engineering of mammalian cells will enable not only sophisticated genetic studies in the context of the genome, but also widely-applicable universal therapeutics based on the pinpointing and correction of the disease-causing genetic elements within the genome in the near future.

  15. Post-Fragmentation Whole Genome Amplification-Based Method

    NASA Technical Reports Server (NTRS)

    Benardini, James; LaDuc, Myron T.; Langmore, John

    2011-01-01

    This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (<400 bp) constitute a library of DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have

  16. Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An annotation-based, genome-wide SNP discovery pipeline is reported using NGS data for large and complex genomes without a reference genome sequence. Roche 454 shotgun reads with low genome coverage of one genotype are annotated in order to distinguish single-copy sequences and repeat junctions fr...

  17. Single Cell Genomics and Transcriptomics for Unicellular Eukaryotes

    SciTech Connect

    Ciobanu, Doina; Clum, Alicia; Singh, Vasanth; Salamov, Asaf; Han, James; Copeland, Alex; Grigoriev, Igor; James, Timothy; Singer, Steven; Woyke, Tanja; Malmstrom, Rex; Cheng, Jan-Fang

    2014-03-14

    Despite their small size, unicellular eukaryotes have complex genomes with a high degree of plasticity that allow them to adapt quickly to environmental changes. Unicellular eukaryotes live with prokaryotes and higher eukaryotes, frequently in symbiotic or parasitic niches. To this day their contribution to the dynamics of the environmental communities remains to be understood. Unfortunately, the vast majority of eukaryotic microorganisms are either uncultured or unculturable, making genome sequencing impossible using traditional approaches. We have developed an approach to isolate unicellular eukaryotes of interest from environmental samples, and to sequence and analyze their genomes and transcriptomes. We have tested our methods with six species: an uncharacterized protist from cellulose-enriched compost identified as Platyophrya, a close relative of P. vorax; the fungus Metschnikowia bicuspidate, a parasite of water flea Daphnia; the mycoparasitic fungi Piptocephalis cylindrospora, a parasite of Cokeromyces and Mucor; Caulochytrium protosteloides, a parasite of Sordaria; Rozella allomycis, a parasite of the water mold Allomyces; and the microalgae Chlamydomonas reinhardtii. Here, we present the four components of our approach: pre-sequencing methods, sequence analysis for single cell genome assembly, sequence analysis of single cell transcriptomes, and genome annotation. This technology has the potential to uncover the complexity of single cell eukaryotes and their role in the environmental samples.

  18. Variation block-based genomics method for crop plants

    PubMed Central

    2014-01-01

    Background In contrast with wild species, cultivated crop genomes consist of reshuffled recombination blocks, which occurred by crossing and selection processes. Accordingly, recombination block-based genomics analysis can be an effective approach for the screening of target loci for agricultural traits. Results We propose the variation block method, which is a three-step process for recombination block detection and comparison. The first step is to detect variations by comparing the short-read DNA sequences of the cultivar to the reference genome of the target crop. Next, sequence blocks with variation patterns are examined and defined. The boundaries between the variation-containing sequence blocks are regarded as recombination sites. All the assumed recombination sites in the cultivar set are used to split the genomes, and the resulting sequence regions are termed variation blocks. Finally, the genomes are compared using the variation blocks. The variation block method identified recurring recombination blocks accurately and successfully represented block-level diversities in the publicly available genomes of 31 soybean and 23 rice accessions. The practicality of this approach was demonstrated by the identification of a putative locus determining soybean hilum color. Conclusions We suggest that the variation block method is an efficient genomics method for the recombination block-level comparison of crop genomes. We expect that this method will facilitate the development of crop genomics by bringing genomics technologies to the field of crop breeding. PMID:24929792

  19. Statistical methods for detecting genomic alterations through array-based comparative genomic hybridization (CGH).

    PubMed

    Wang, Yuedong; Guo, Sun-Wei

    2004-01-01

    Array-based comparative genomic hybridization (ABCGH) is an emerging high-resolution and high-throughput molecular genetic technique that allows genome-wide screening for chromosome alterations associated with tumorigenesis. Like the cDNA microarrays, ABCGH uses two differentially labeled test and reference DNAs which are cohybridized to cloned genomic fragments immobilized on glass slides. The hybridized DNAs are then detected in two different fluorochromes, and the significant deviation from unity in the ratios of the digitized intensity values is indicative of copy-number differences between the test and reference genomes. Proper statistical analyses need to account for many sources of variation besides genuine differences between the two genomes. In particular, spatial correlations, the variable nature of the ratio variance and non-Normal distribution call for careful statistical modeling. We propose two new statistics, the standard t-statistic and its modification with variances smoothed along the genome, and two tests for each statistic, the standard t-test and a test based on the hybrid adaptive spline (HAS). Simulations indicate that the smoothed t-statistic always improves the performance over the standard t-statistic. The t-tests are more powerful in detecting isolated alterations while those based on HAS are more powerful in detecting a cluster of alterations. We apply the proposed methods to the identification of genomic alterations in endometrium in women with endometriosis.

  20. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting | Office of Cancer Genomics

    Cancer.gov

    The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest.

  1. Single-Cell (Meta-)Genomics of a Dimorphic Candidatus Thiomargarita nelsonii Reveals Genomic Plasticity

    PubMed Central

    Flood, Beverly E.; Fliss, Palmer; Jones, Daniel S.; Dick, Gregory J.; Jain, Sunit; Kaster, Anne-Kristin; Winkel, Matthias; Mußmann, Marc; Bailey, Jake

    2016-01-01

    The genus Thiomargarita includes the world's largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group

  2. Genomic Typing of Red Cell Antigens

    DTIC Science & Technology

    2011-09-01

    Antigen‐Matched  Red  Cells   for  Sickle   Cell   Anemia  Patients  Using  Molecular Typing to Augment Testing: Meghan Delaney, Prashant Gaur, Askale...H, Constans J, Quilici JC, Lefevre‐Witier P, Sevin J, Stevens M: Study of red blood  cell  and serum enzymes in  five  Pyrenean communities and in a...Antigen‐Matched Red  Cells  for  Sickle   Cell  Anemia Patients  Using Molecular Typing to Augment Testing: AABB (poster) 2009.  Background: Patients with  sickle

  3. Genome-Scale Analysis of Cell-Specific Regulatory Codes Using Nuclear Enzymes.

    PubMed

    Baek, Songjoon; Sung, Myong-Hee

    2016-01-01

    High-throughput sequencing technologies have made it possible for biologists to generate genome-wide profiles of chromatin features at the nucleotide resolution. Enzymes such as nucleases or transposes have been instrumental as a chromatin-probing agent due to their ability to target accessible chromatin for cleavage or insertion. On the scale of a few hundred base pairs, preferential action of the nuclear enzymes on accessible chromatin allows mapping of cell state-specific accessibility in vivo. Such accessible regions contain functionally important regulatory sites, including promoters and enhancers, which undergo active remodeling for cells adapting in a dynamic environment. DNase-seq and the more recent ATAC-seq are two assays that are gaining popularity. Deep sequencing of DNA libraries from these assays, termed genomic footprinting, has been proposed to enable the comprehensive construction of protein occupancy profiles over the genome at the nucleotide level. Recent studies have discovered limitations of genomic footprinting which reduce the scope of detectable proteins. In addition, the identification of putative factors that bind to the observed footprints remains challenging. Despite these caveats, the methodology still presents significant advantages over alternative techniques such as ChIP-seq or FAIRE-seq. Here we describe computational approaches and tools for analysis of chromatin accessibility and genomic footprinting. Proper experimental design and assay-specific data analysis ensure the detection sensitivity and maximize retrievable information. The enzyme-based chromatin profiling approaches represent a powerful and evolving methodology which facilitates our understanding of how the genome is regulated.

  4. [Transcription activator-like effectors(TALEs)based genome engineering].

    PubMed

    Zhao, Mei-Wei; Duan, Cheng-Li; Liu, Jiang

    2013-10-01

    Systematic reverse-engineering of functional genome architecture requires precise modifications of gene sequences and transcription levels. The development and application of transcription activator-like effectors(TALEs) has created a wealth of genome engineering possibilities. TALEs are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas species. The DNA-binding domain of each TALE typically consists of tandem 34-amino acid repeat modules rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Such "genome engineering" has now been established in human cells and a number of model organisms, thus opening the door to better understanding gene function in model organisms, improving traits in crop plants and treating human genetic disorders.

  5. Programming the genome in embryonic and somatic stem cells.

    PubMed

    Collas, Philippe; Noer, Agate; Timoskainen, Sanna

    2007-01-01

    In opposition to terminally differentiated cells, stem cells can self-renew and give rise to multiple cell types. Embryonic stem cells retain the ability of the inner cell mass of blastocysts to differentiate into all cell types of the body and have acquired in culture unlimited self-renewal capacity. Somatic stem cells are found in many adult tissues, have an extensive but finite lifespan and can differentiate into a more restricted array of cell types. A growing body of evidence indicates that multi-lineage differentiation ability of stem cells can be defined by the potential for expression of lineage-specification genes. Gene expression, or as emphasized here, potential for gene expression, is largely controlled by epigenetic modifications of DNA and chromatin on genomic regulatory and coding regions. These modifications modulate chromatin organization not only on specific genes but also at the level of the whole nucleus; they can also affect timing of DNA replication. This review highlights how mechanisms by which genes are poised for transcription in undifferentiated stem cells are being uncovered through primarily the mapping of DNA methylation, histone modifications and transcription factor binding throughout the genome. The combinatorial association of epigenetic marks on developmentally regulated and lineage-specifying genes in undifferentiated cells seems to define a pluripotent state.

  6. Base-Resolution Analysis of Cisplatin–DNA Adducts at the Genome Scale

    PubMed Central

    Shu, Xiaoting; Xiong, Xushen; Song, Jinghui; He, Chuan; Yi, Chengqi

    2016-01-01

    Cisplatin, one of the most widely used anticancer drugs, crosslinks DNA and ultimately induces cell death. However, the genomic pattern of cisplatin–DNA adducts has remained unknown owing to the lack of a reliable and sensitive genome-wide method. Herein we present “cisplatin-seq” to identify genome-wide cisplatin crosslinking sites at base resolution. Cisplatin-seq reveals that mitochondrial DNA is a preferred target of cisplatin. For nuclear genomes, cisplatin–DNA adducts are enriched within promoters and regions harboring transcription termination sites. While the density of GG dinucleotides determines the initial crosslinking of cisplatin, binding of proteins to the genome largely contributes to the accumulative pattern of cisplatin–DNA adducts. PMID:27736024

  7. SpBase: the sea urchin genome database and web site.

    PubMed

    Cameron, R Andrew; Samanta, Manoj; Yuan, Autumn; He, Dong; Davidson, Eric

    2009-01-01

    SpBase is a system of databases focused on the genomic information from sea urchins and related echinoderms. It is exposed to the public through a web site served with open source software (http://spbase.org/). The enterprise was undertaken to provide an easily used collection of information to directly support experimental work on these useful research models in cell and developmental biology. The information served from the databases emerges from the draft genomic sequence of the purple sea urchin, Strongylocentrotus purpuratus and includes sequence data and genomic resource descriptions for other members of the echinoderm clade which in total span 540 million years of evolutionary time. This version of the system contains two assemblies of the purple sea urchin genome, associated expressed sequences, gene annotations and accessory resources. Search mechanisms for the sequences and the gene annotations are provided. Because the system is maintained along with the Sea Urchin Genome resource, a database of sequenced clones is also provided.

  8. CGCI Investigators Reveal Comprehensive Landscape of Diffuse Large B-Cell Lymphoma (DLBCL) Genomes | Office of Cancer Genomics

    Cancer.gov

    Researchers from British Columbia Cancer Agency used whole genome sequencing to analyze 40 DLBCL cases and 13 cell lines in order to fill in the gaps of the complex landscape of DLBCL genomes. Their analysis, “Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing,” was published online in Blood on May 22. The authors are Ryan Morin, Marco Marra, and colleagues.  

  9. Genome of a Low-Salinity Ammonia-Oxidizing Archaeon Determined by Single-Cell and Metagenomic Analysis

    PubMed Central

    Potanina, Anastasia; Francis, Christopher A.; Quake, Stephen R.

    2011-01-01

    Ammonia-oxidizing archaea (AOA) are thought to be among the most abundant microorganisms on Earth and may significantly impact the global nitrogen and carbon cycles. We sequenced the genome of AOA in an enrichment culture from low-salinity sediments in San Francisco Bay using single-cell and metagenomic genome sequence data. Five single cells were isolated inside an integrated microfluidic device using laser tweezers, the cells' genomic DNA was amplified by multiple displacement amplification (MDA) in 50 nL volumes and then sequenced by high-throughput DNA pyrosequencing. This microscopy-based approach to single-cell genomics minimizes contamination and allows correlation of high-resolution cell images with genomic sequences. Statistical properties of coverage across the five single cells, in combination with the contrasting properties of the metagenomic dataset allowed the assembly of a high-quality draft genome. The genome of this AOA, which we designate Candidatus Nitrosoarchaeum limnia SFB1, is ∼1.77 Mb with >2100 genes and a G+C content of 32%. Across the entire genome, the average nucleotide identity to Nitrosopumilus maritimus, the only AOA in pure culture, is ∼70%, suggesting this AOA represents a new genus of Crenarchaeota. Phylogenetically, the 16S rRNA and ammonia monooxygenase subunit A (amoA) genes of this AOA are most closely related to sequences reported from a wide variety of freshwater ecosystems. Like N. maritimus, the low-salinity AOA genome appears to have an ammonia oxidation pathway distinct from ammonia oxidizing bacteria (AOB). In contrast to other described AOA, these low-salinity AOA appear to be motile, based on the presence of numerous motility- and chemotaxis-associated genes in the genome. This genome data will be used to inform targeted physiological and metabolic studies of this novel group of AOA, which may ultimately advance our understanding of AOA metabolism and their impacts on the global carbon and nitrogen cycles. PMID

  10. Atypical Epstein-Barr viral genomic structure in lymphoma tissue and lymphoid cell lines.

    PubMed

    Tang, Weihua; Fan, Hongxin; Schroeder, Jane; Dunphy, Cherie H; Bryant, Ronald J; Fedoriw, Yuri; Gulley, Margaret L

    2013-06-01

    Epstein-Barr virus (EBV) DNA is found within the malignant cells of some subtypes of lymphoma, and viral presence is being exploited for improved diagnosis, monitoring, and management of affected patients. Recent work suggests that viral genomic polymorphism, such as partial deletion of the viral genome, could interfere with virus detection in tumor tissues. To test for atypical forms of the EBV genome, 98 lymphomas and 6 infected cell lines were studied using a battery of 6 quantitative polymerase chain reaction assays targeting disparate sections of EBV DNA. Fifty of the lymphomas (51%) had no amplifiable EBV DNA, and 38 lymphomas (39%) had low-level EBV infection that was deemed incidental based on EBV-encoded RNA (EBER) in situ hybridization results. The remaining 10 lymphomas (10%) had high EBV loads and EBER localization to malignant cells by EBER in situ hybridization. All 10 represented lymphoma subtypes were previously associated with EBV (Burkitt, diffuse large B-cell, or T-cell type), whereas no remnants of EBV were detected in other lymphoma subtypes (follicular, small lymphocytic, mantle cell, or marginal zone type). Interestingly, 4 of the 10 infected lymphomas had evidence of atypical viral genomes, including 3 of 4 infected T-cell lymphomas with aberrant loss of LMP2 amplicons, and a single diffuse large B-cell lymphoma lacking the central part of the viral genome spanning BamH1W, BZLF1, and EBNA1 gene segments. A reasonable screening strategy for infected malignancy involves applying EBER1 and LMP1 quantitative polymerase chain reaction assays and confirming that values exceeding 2000 copies of EBV per 100,000 cells have EBER localization to malignant cells.

  11. Two ways to fold the genome during the cell cycle: insights obtained with chromosome conformation capture.

    PubMed

    Dekker, Job

    2014-01-01

    Genetic and epigenetic inheritance through mitosis is critical for dividing cells to maintain their state. This process occurs in the context of large-scale re-organization of chromosome conformation during prophase leading to the formation of mitotic chromosomes, and during the reformation of the interphase nucleus during telophase and early G1. This review highlights how recent studies over the last 5 years employing chromosome conformation capture combined with classical models of chromosome organization based on decades of microscopic observations, are providing new insights into the three-dimensional organization of chromatin inside the interphase nucleus and within mitotic chromosomes. One striking observation is that interphase genome organization displays cell type-specific features that are related to cell type-specific gene expression, whereas mitotic chromosome folding appears universal and tissue invariant. This raises the question of whether or not there is a need for an epigenetic memory for genome folding. Herein, the two different folding states of mammalian genomes are reviewed and then models are discussed wherein instructions for cell type-specific genome folding are locally encoded in the linear genome and transmitted through mitosis, e.g., as open chromatin sites with or without continuous binding of transcription factors. In the next cell cycle these instructions are used to re-assemble protein complexes on regulatory elements which then drive three-dimensional folding of the genome from the bottom up through local action and self-assembly into higher order levels of cell type-specific organization. In this model, no explicit epigenetic memory for cell type-specific chromosome folding is required.

  12. Detection of aneuploidy in single cells using comparative genomic hybridization.

    PubMed

    Voullaire, L; Wilton, L; Slater, H; Williamson, R

    1999-09-01

    The ability of comparative genomic hybridization (CGH) to detect aneuploidy following universal amplification of DNA from a single cell, or a small number of cells, was investigated with a view to preimplantation diagnosis following in vitro fertilization, and prenatal diagnosis using fetal erythroblasts obtained from maternal blood. The DNA obtained from lysed single cells was amplified using degenerate oligonucleotide-primed PCR (DOP-PCR). This product was labelled using nick translation and hybridized together with normal reference genomic DNA. The CGH fluorescent ratio profiles obtained could be used to determine aneuploidy with cut-off thresholds of 0.75 and 1.25. Deviation in the profiles in the heterochromatic regions was reduced by using, as a reference sample, normal genomic DNA that had also undergone DOP-PCR. Single cells known to be trisomic for chromosomes 13, 18 or 21 were analysed using this technique. The resolution of CGH with amplified DNA from a single cell is of the order of 40 Mb, sufficient for the diagnosis of trisomy 21, and possibly segmental aneuploidy of equivalent size. These results, and those of others, demonstrate that diagnosis of chromosomal aneuploidy in single cells is possible using CGH with DOP-PCR amplified DNA.

  13. Programming cells by multiplex genome engineering and accelerated evolution

    PubMed Central

    Carr, Peter A.; Sun, Zachary Z.; Xu, George; Forest, Craig R.; Church, George M.

    2015-01-01

    The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments1,2. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales3. Although in vitro and directed evolution methods4–9 have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-d-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties. PMID:19633652

  14. Induced Pluripotent Stem Cells Meet Genome Editing.

    PubMed

    Hockemeyer, Dirk; Jaenisch, Rudolf

    2016-05-05

    It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments-the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success.

  15. Exploring Arabidopsis thaliana Root Endophytes via Single-Cell Genomics

    SciTech Connect

    Lundberg, Derek; Woyke, Tanja; Tringe, Susannah; Dangl, Jeff

    2014-03-19

    Land plants grow in association with microbial communities both on their surfaces and inside the plant (endophytes). The relationships between microbes and their host can vary from pathogenic to mutualistic. Colonization of the endophyte compartment occurs in the presence of a sophisticated plant immune system, implying finely tuned discrimination of pathogens from mutualists and commensals. Despite the importance of the microbiome to the plant, relatively little is known about the specific interactions between plants and microbes, especially in the case of endophytes. The vast majority of microbes have not been grown in the lab, and thus one of the few ways of studying them is by examining their DNA. Although metagenomics is a powerful tool for examining microbial communities, its application to endophyte samples is technically difficult due to the presence of large amounts of host plant DNA in the sample. One method to address these difficulties is single-cell genomics where a single microbial cell is isolated from a sample, lysed, and its genome amplified by multiple displacement amplification (MDA) to produce enough DNA for genome sequencing. This produces a single-cell amplified genome (SAG). We have applied this technology to study the endophytic microbes in Arabidopsis thaliana roots. Extensive 16S gene profiling of the microbial communities in the roots of multiple inbred A. thaliana strains has identified 164 OTUs as being significantly enriched in all the root endophyte samples compared to their presence in bulk soil.

  16. Effects of whole genome duplication on cell size and gene expression in mouse embryonic stem cells

    PubMed Central

    IMAI, Hiroyuki; FUJII, Wataru; KUSAKABE, Ken Takeshi; KISO, Yasuo; KANO, Kiyoshi

    2016-01-01

    Alterations in ploidy tend to influence cell physiology, which in the long-term, contribute to species adaptation and evolution. Polyploid cells are observed under physiological conditions in the nerve and liver tissues, and in tumorigenic processes. Although tetraploid cells have been studied in mammalian cells, the basic characteristics and alterations caused by whole genome duplication are still poorly understood. The purpose of this study was to acquire basic knowledge about the effect of whole genome duplication on the cell cycle, cell size, and gene expression. Using flow cytometry, we demonstrate that cell cycle subpopulations in mouse tetraploid embryonic stem cells (TESCs) were similar to those in embryonic stem cells (ESCs). We performed smear preparations and flow cytometric analysis to identify cell size alterations. These indicated that the relative cell volume of TESCs was approximately 2.2–2.5 fold that of ESCs. We also investigated the effect of whole genome duplication on the expression of housekeeping and pluripotency marker genes using quantitative real-time PCR with external RNA. We found that the target transcripts were 2.2 times more abundant in TESCs than those in ESCs. This indicated that gene expression and cell volume increased in parallel. Our findings suggest the existence of a homeostatic mechanism controlling the cytoplasmic transcript levels in accordance with genome volume changes caused by whole genome duplication. PMID:27569766

  17. Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

    PubMed Central

    Wang, Ling; Yang, Likai; Guo, Yijie; Du, Weili; Yin, Yajun; Zhang, Tao; Lu, Hongzhao

    2017-01-01

    The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms. PMID:28068387

  18. A Method for Checking Genomic Integrity in Cultured Cell Lines from SNP Genotyping Data.

    PubMed

    Danecek, Petr; McCarthy, Shane A; Durbin, Richard

    2016-01-01

    Genomic screening for chromosomal abnormalities is an important part of quality control when establishing and maintaining stem cell lines. We present a new method for sensitive detection of copy number alterations, aneuploidy, and contamination in cell lines using genome-wide SNP genotyping data. In contrast to other methods designed for identifying copy number variations in a single sample or in a sample composed of a mixture of normal and tumor cells, this new method is tailored for determining differences between cell lines and the starting material from which they were derived, which allows us to distinguish between normal and novel copy number variation. We implemented the method in the freely available BCFtools package and present results based on induced pluripotent stem cell lines obtained in the HipSci project.

  19. A Method for Checking Genomic Integrity in Cultured Cell Lines from SNP Genotyping Data

    PubMed Central

    McCarthy, Shane A.; Durbin, Richard

    2016-01-01

    Genomic screening for chromosomal abnormalities is an important part of quality control when establishing and maintaining stem cell lines. We present a new method for sensitive detection of copy number alterations, aneuploidy, and contamination in cell lines using genome-wide SNP genotyping data. In contrast to other methods designed for identifying copy number variations in a single sample or in a sample composed of a mixture of normal and tumor cells, this new method is tailored for determining differences between cell lines and the starting material from which they were derived, which allows us to distinguish between normal and novel copy number variation. We implemented the method in the freely available BCFtools package and present results based on induced pluripotent stem cell lines obtained in the HipSci project. PMID:27176002

  20. Genome Editing in Human Cells Using CRISPR/Cas Nucleases.

    PubMed

    Wyvekens, Nicolas; Tsai, Shengdar Q; Joung, J Keith

    2015-10-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. This unit describes protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases.

  1. RNA-programmed genome editing in human cells.

    PubMed

    Jinek, Martin; East, Alexandra; Cheng, Aaron; Lin, Steven; Ma, Enbo; Doudna, Jennifer

    2013-01-29

    Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3' end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.DOI:http://dx.doi.org/10.7554/eLife.00471.001.

  2. Current Developments in Prokaryotic Single Cell Whole Genome Amplification

    SciTech Connect

    Goudeau, Danielle; Nath, Nandita; Ciobanu, Doina; Cheng, Jan-Fang; Malmstrom, Rex

    2014-03-14

    Our approach to prokaryotic single-cell Whole Genome Amplification at the JGI continues to evolve. To increase both the quality and number of single-cell genomes produced, we explore all aspects of the process from cell sorting to sequencing. For example, we now utilize specialized reagents, acoustic liquid handling, and reduced reaction volumes eliminate non-target DNA contamination in WGA reactions. More specifically, we use a cleaner commercial WGA kit from Qiagen that employs a UV decontamination procedure initially developed at the JGI, and we use the Labcyte Echo for tip-less liquid transfer to set up 2uL reactions. Acoustic liquid handling also dramatically reduces reagent costs. In addition, we are exploring new cell lysis methods including treatment with Proteinase K, lysozyme, and other detergents, in order to complement standard alkaline lysis and allow for more efficient disruption of a wider range of cells. Incomplete lysis represents a major hurdle for WGA on some environmental samples, especially rhizosphere, peatland, and other soils. Finding effective lysis strategies that are also compatible with WGA is challenging, and we are currently assessing the impact of various strategies on genome recovery.

  3. The somatic genomic landscape of chromophobe renal cell carcinoma.

    PubMed

    Davis, Caleb F; Ricketts, Christopher J; Wang, Min; Yang, Lixing; Cherniack, Andrew D; Shen, Hui; Buhay, Christian; Kang, Hyojin; Kim, Sang Cheol; Fahey, Catherine C; Hacker, Kathryn E; Bhanot, Gyan; Gordenin, Dmitry A; Chu, Andy; Gunaratne, Preethi H; Biehl, Michael; Seth, Sahil; Kaipparettu, Benny A; Bristow, Christopher A; Donehower, Lawrence A; Wallen, Eric M; Smith, Angela B; Tickoo, Satish K; Tamboli, Pheroze; Reuter, Victor; Schmidt, Laura S; Hsieh, James J; Choueiri, Toni K; Hakimi, A Ari; Chin, Lynda; Meyerson, Matthew; Kucherlapati, Raju; Park, Woong-Yang; Robertson, A Gordon; Laird, Peter W; Henske, Elizabeth P; Kwiatkowski, David J; Park, Peter J; Morgan, Margaret; Shuch, Brian; Muzny, Donna; Wheeler, David A; Linehan, W Marston; Gibbs, Richard A; Rathmell, W Kimryn; Creighton, Chad J

    2014-09-08

    We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) on the basis of multidimensional and comprehensive characterization, including mtDNA and whole-genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared with other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT upregulation in cancer distinct from previously observed amplifications and point mutations.

  4. A Web-Based Comparative Genomics Tutorial for Investigating Microbial Genomes

    PubMed Central

    STRONG, MICHAEL; CASCIO, DUILIO; EISENBERG, DAVID

    2004-01-01

    As the number of completely sequenced microbial genomes continues to rise at an impressive rate, it is important to prepare students with the skills necessary to investigate microorganisms at the genomic level. As a part of the core curriculum for first-year graduate students in the biological sciences, we have implemented a web-based tutorial to introduce students to the fields of comparative and functional genomics. The tutorial focuses on recent computational methods for identifying functionally linked genes and proteins on a genome-wide scale and was used to introduce students to the Rosetta Stone, Phylogenetic Profile, conserved Gene Neighbor, and Operon computational methods. Students learned to use a number of publicly available web servers and databases to identify functionally linked genes in the Escherichia coli genome, with emphasis on genome organization and operon structure. The overall effectiveness of the tutorial was assessed based on student evaluations and homework assignments. The tutorial is available to other educators at http://www.doe-mbi.ucla.edu/~strong/m253.php. PMID:23653555

  5. Comprehensive genomic characterization of squamous cell lung cancers

    PubMed Central

    2012-01-01

    Summary Lung squamous cell carcinoma (lung SqCC) is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in lung SqCC have not been comprehensively characterized and no molecularly targeted agents have been developed specifically for its treatment. As part of The Cancer Genome Atlas (TCGA), we profiled 178 lung SqCCs to provide a comprehensive landscape of genomic and epigenomic alterations. Lung SqCC is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumor. We found statistically recurrent mutations in 18 genes in including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations were seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2/KEAP1 in 34%, squamous differentiation genes in 44%, PI3K/AKT in 47%, and CDKN2A/RB1 in 72% of tumors. We identified a potential therapeutic target in the majority of tumors, offering new avenues of investigation for lung SqCC treatment. PMID:22960745

  6. KRAS Genomic Status Predicts the Sensitivity of Ovarian Cancer Cells to Decitabine | Office of Cancer Genomics

    Cancer.gov

    Decitabine, a cancer therapeutic that inhibits DNA methylation, produces variable antitumor response rates in patients with solid tumors that might be leveraged clinically with identification of a predictive biomarker. In this study, we profiled the response of human ovarian, melanoma, and breast cancer cells treated with decitabine, finding that RAS/MEK/ERK pathway activation and DNMT1 expression correlated with cytotoxic activity. Further, we showed that KRAS genomic status predicted decitabine sensitivity in low-grade and high-grade serous ovarian cancer cells.

  7. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

    PubMed Central

    Clark, Joseph T; Kennedy, Andrew B; Ryan, Daniel E; Roy, Subhadeep; Steinfeld, Israel; Lunstad, Benjamin D; Kaiser, Robert J; Wilkens, Alec B; Bacchetta, Rosa; Tsalenko, Anya; Dellinger, Douglas; Bruhn, Laurakay; Porteus, Matthew H

    2016-01-01

    CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology. PMID:26121415

  8. Architectural proteins: regulators of 3D genome organization in cell fate.

    PubMed

    Gómez-Díaz, Elena; Corces, Victor G

    2014-11-01

    The relation between alterations in chromatin structure and changes in gene expression during cell differentiation has served as a paradigm to understand the link between genome organization and function. Yet, the factors involved and the mechanisms by which the 3D organization of the nucleus is established remain poorly understood. The use of Chromosome Conformation-Capture (3C)-based approaches has resulted in a new appreciation of the role of architectural proteins in the establishment of 3D genome organization. Architectural proteins orchestrate higher-order chromatin organization through the establishment of interactions between regulatory elements across multiple spatial scales. The regulation of these proteins, their interaction with DNA, and their co-occurrence in the genome, may be responsible for the plasticity of 3D chromatin architecture that dictates cell and time-specific blueprints of gene expression.

  9. Architectural proteins: Regulators of 3D genome organization in cell fate

    PubMed Central

    Gómez-Díaz, Elena; Corces, Victor G.

    2014-01-01

    The relationship between alterations in chromatin structure and changes in gene expression during cell differentiation has served as a paradigm to understand the link between genome organization and function. Yet the factors involved and the mechanisms by which the three-dimensional organization of the nucleus is established remain poorly understood. The use of Chromosome Conformation-Capture (3C) based approaches has resulted in a new appreciation of the role of architectural proteins in the establishment of 3D genome organization. Architectural proteins orchestrate higher-order chromatin organization through the establishment of interactions between regulatory elements across multiple spatial scales. The regulation of these proteins, their interaction with DNA, and their co occurrence in the genome, may be responsible for the plasticity of 3D-chromatin architecture that dictates cell and time-specific blueprints of gene expression. PMID:25218583

  10. [Compartmentalization of the cell nucleus and spatial organization of the genome].

    PubMed

    Gavrilov, A A; Razin, S V

    2015-01-01

    The eukaryotic cell nucleus is one of the most complex cell organelles. Despite the absence of membranes, the nuclear space is divided into numerous compartments where different processes in- volved in the genome activity take place. The most important nuclear compartments include nucleoli, nuclear speckles, PML bodies, Cajal bodies, histone locus bodies, Polycomb bodies, insulator bodies, transcription and replication factories. The structural basis for the nuclear compartmentalization is provided by genomic DNA that occupies most of the nuclear volume. Nuclear compartments, in turn, guide the chromosome folding by providing a platform for the spatial interaction of individual genomic loci. In this review, we discuss fundamental principles of higher order genome organization with a focus on chromosome territories and chromosome domains, as well as consider the structure and function of the key nuclear compartments. We show that the func- tional compartmentalization of the cell nucleus and genome spatial organization are tightly interconnected, and that this form of organization is highly dynamic and is based on stochastic processes.

  11. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines

    PubMed Central

    Contino, Gianmarco; Eldridge, Matthew D.; Secrier, Maria; Bower, Lawrence; Fels Elliott, Rachael; Weaver, Jamie; Lynch, Andy G.; Edwards, Paul A.W.; Fitzgerald, Rebecca C.

    2016-01-01

    Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC. PMID:27594985

  12. Modeling genome coverage in single-cell sequencing

    PubMed Central

    Daley, Timothy; Smith, Andrew D.

    2014-01-01

    Motivation: Single-cell DNA sequencing is necessary for examining genetic variation at the cellular level, which remains hidden in bulk sequencing experiments. But because they begin with such small amounts of starting material, the amount of information that is obtained from single-cell sequencing experiment is highly sensitive to the choice of protocol employed and variability in library preparation. In particular, the fraction of the genome represented in single-cell sequencing libraries exhibits extreme variability due to quantitative biases in amplification and loss of genetic material. Results: We propose a method to predict the genome coverage of a deep sequencing experiment using information from an initial shallow sequencing experiment mapped to a reference genome. The observed coverage statistics are used in a non-parametric empirical Bayes Poisson model to estimate the gain in coverage from deeper sequencing. This approach allows researchers to know statistical features of deep sequencing experiments without actually sequencing deeply, providing a basis for optimizing and comparing single-cell sequencing protocols or screening libraries. Availability and implementation: The method is available as part of the preseq software package. Source code is available at http://smithlabresearch.org/preseq. Contact: andrewds@usc.edu Supplementary information: Supplementary material is available at Bioinformatics online. PMID:25107873

  13. Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis

    SciTech Connect

    Utturkar, Sagar M.; Cude, W. Nathan; Robeson, Jr., Michael S.; Yang, Zamin Koo; Klingeman, Dawn Marie; Land, Miriam L.; Allman, Steve L.; Lu, Tse-Yuan S.; Brown, Steven D.; Schadt, Christopher Warren; Podar, Mircea; Doktycz, Mitchel J.; Pelletier, Dale A.

    2016-07-15

    Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. In this paper, we present a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Finally, comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.

  14. Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis

    DOE PAGES

    Utturkar, Sagar M.; Cude, W. Nathan; Robeson, Jr., Michael S.; ...

    2016-07-15

    Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. In this paper, we present a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from themore » plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Finally, comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.« less

  15. Detection of genomic imbalances by array based comparative genomic hybridisation in fetuses with multiple malformations

    PubMed Central

    Le Caignec, C; Boceno, M; Saugier-Veber, P; Jacquemont, S; Joubert, M; David, A; Frebourg, T; Rival, J

    2005-01-01

    Background: Malformations are a major cause of morbidity and mortality in full term infants and genomic imbalances are a significant component of their aetiology. However, the causes of defects in many patients with multiple congenital malformations remain unexplained despite thorough clinical examination and laboratory investigations. Methods: We used a commercially available array based comparative genomic hybridisation method (array CGH), able to screen all subtelomeric regions, main microdeletion syndromes, and 201 other regions covering the genome, to detect submicroscopic chromosomal imbalances in 49 fetuses with three or more significant anomalies and normal karyotype. Results: Array CGH identified eight genomic rearrangements (16.3%), all confirmed by quantitative multiplex PCR of short fluorescent fragments. Subtelomeric and interstitial deletions, submicroscopic duplications, and a complex genomic imbalance were identified. In four de novo cases (15qtel deletion, 16q23.1–q23.3 deletion, 22q11.2 deletion, and mosaicism for a rearranged chromosome 18), the genomic imbalance identified clearly underlay the pathological phenotype. In one case, the relationship between the genotype and phenotype was unclear, since a subtelomeric 6q deletion was detected in a mother and her two fetuses bearing multiple malformations. In three cases, a subtelomeric 10q duplication, probably a genomic polymorphism, was identified. Conclusions: The detection of 5/49 causative chromosomal imbalances (or 4/49 if the 6qtel deletion is not considered as causative) suggests wide genome screening when standard chromosome analysis is normal and confirms that array CGH will have a major impact on pre and postnatal diagnosis as well as providing information for more accurate genetic counselling. PMID:15689449

  16. OMIGA: Optimized Maker-Based Insect Genome Annotation.

    PubMed

    Liu, Jinding; Xiao, Huamei; Huang, Shuiqing; Li, Fei

    2014-08-01

    Insects are one of the largest classes of animals on Earth and constitute more than half of all living species. The i5k initiative has begun sequencing of more than 5,000 insect genomes, which should greatly help in exploring insect resource and pest control. Insect genome annotation remains challenging because many insects have high levels of heterozygosity. To improve the quality of insect genome annotation, we developed a pipeline, named Optimized Maker-Based Insect Genome Annotation (OMIGA), to predict protein-coding genes from insect genomes. We first mapped RNA-Seq reads to genomic scaffolds to determine transcribed regions using Bowtie, and the putative transcripts were assembled using Cufflink. We then selected highly reliable transcripts with intact coding sequences to train de novo gene prediction software, including Augustus. The re-trained software was used to predict genes from insect genomes. Exonerate was used to refine gene structure and to determine near exact exon/intron boundary in the genome. Finally, we used the software Maker to integrate data from RNA-Seq, de novo gene prediction, and protein alignment to produce an official gene set. The OMIGA pipeline was used to annotate the draft genome of an important insect pest, Chilo suppressalis, yielding 12,548 genes. Different strategies were compared, which demonstrated that OMIGA had the best performance. In summary, we present a comprehensive pipeline for identifying genes in insect genomes that can be widely used to improve the annotation quality in insects. OMIGA is provided at http://ento.njau.edu.cn/omiga.html .

  17. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

  18. Comprehensive genome characterization of solitary fibrous tumors using high-resolution array-based comparative genomic hybridization.

    PubMed

    Bertucci, François; Bouvier-Labit, Corinne; Finetti, Pascal; Adélaïde, José; Metellus, Philippe; Mokhtari, Karima; Decouvelaere, Anne-Valérie; Miquel, Catherine; Jouvet, Anne; Figarella-Branger, Dominique; Pedeutour, Florence; Chaffanet, Max; Birnbaum, Daniel

    2013-02-01

    Solitary fibrous tumors (SFTs) are rare spindle cell tumors with limited therapeutic options. Their molecular basis is poorly known. No consistent cytogenetic abnormality has been reported. We used high-resolution whole-genome array-based comparative genomic hybridization (Agilent 244K oligonucleotide chips) to profile 47 samples, meningeal in >75% of cases. Few copy number aberrations (CNAs) were observed. Sixty-eight percent of samples did not show any gene CNA after exclusion of probes located in regions with referenced copy number variation (CNV). Only low-level CNAs were observed. The genomic profiles were very homogeneous among samples. No molecular class was revealed by clustering of DNA copy numbers. All cases displayed a "simplex" profile. No recurrent CNA was identified. Imbalances occurring in >20%, such as the gain of 8p11.23-11.22 region, contained known CNVs. The 13q14.11-13q31.1 region (lost in 4% of cases) was the largest altered region and contained the lowest percentage of genes with referenced CNVs. A total of 425 genes without CNV showed copy number transition in at least one sample, but only but only 1 in at least 10% of samples. The genomic profiles of meningeal and extra-meningeal cases did not show any differences.

  19. MarinegenomicsDB: an integrated genome viewer for community-based annotation of genomes.

    PubMed

    Koyanagi, Ryo; Takeuchi, Takeshi; Hisata, Kanako; Gyoja, Fuki; Shoguchi, Eiichi; Satoh, Nori; Kawashima, Takeshi

    2013-10-01

    We constructed a web-based genome annotation platform, MarinegenomicsDB, to integrate genome data from various marine organisms including the pearl oyster Pinctada fucata and the coral Acropora digitifera. This newly developed viewer application provides open access to published data and a user-friendly environment for community-based manual gene annotation. Development on a flexible framework enables easy expansion of the website on demand. To date, more than 2000 genes have been annotated using this system. In the future, the website will be expanded to host a wider variety of data, more species, and different types of genome-wide analyses. The website is available at the following URL: http://marinegenomics.oist.jp.

  20. CnidBase: The Cnidarian Evolutionary Genomics Database

    PubMed Central

    Ryan, Joseph F.; Finnerty, John R.

    2003-01-01

    CnidBase, the Cnidarian Evolutionary Genomics Database, is a tool for investigating the evolutionary, developmental and ecological factors that affect gene expression and gene function in cnidarians. In turn, CnidBase will help to illuminate the role of specific genes in shaping cnidarian biodiversity in the present day and in the distant past. CnidBase highlights evolutionary changes between species within the phylum Cnidaria and structures genomic and expression data to facilitate comparisons to non-cnidarian metazoans. CnidBase aims to further the progress that has already been made in the realm of cnidarian evolutionary genomics by creating a central community resource which will help drive future research and facilitate more accurate classification and comparison of new experimental data with existing data. CnidBase is available at http://cnidbase.bu.edu/. PMID:12519972

  1. Genomic imprinting in development, growth, behavior and stem cells.

    PubMed

    Plasschaert, Robert N; Bartolomei, Marisa S

    2014-05-01

    Genes that are subject to genomic imprinting in mammals are preferentially expressed from a single parental allele. This imprinted expression of a small number of genes is crucial for normal development, as these genes often directly regulate fetal growth. Recent work has also demonstrated intricate roles for imprinted genes in the brain, with important consequences on behavior and neuronal function. Finally, new studies have revealed the importance of proper expression of specific imprinted genes in induced pluripotent stem cells and in adult stem cells. As we review here, these findings highlight the complex nature and developmental importance of imprinted genes.

  2. Mechanical Genomics Identifies Diverse Modulators of Bacterial Cell Stiffness.

    PubMed

    Auer, George K; Lee, Timothy K; Rajendram, Manohary; Cesar, Spencer; Miguel, Amanda; Huang, Kerwyn Casey; Weibel, Douglas B

    2016-06-22

    Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of ∼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted. We observe that proteins with biochemically redundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducing membrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall and broader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.

  3. Small cell ovarian carcinoma: genomic stability and responsiveness to therapeutics

    PubMed Central

    2013-01-01

    Background The biology of small cell ovarian carcinoma of the hypercalcemic type (SCCOHT), which is a rare and aggressive form of ovarian cancer, is poorly understood. Tumourigenicity, in vitro growth characteristics, genetic and genomic anomalies, and sensitivity to standard and novel chemotherapeutic treatments were investigated in the unique SCCOHT cell line, BIN-67, to provide further insight in the biology of this rare type of ovarian cancer. Method The tumourigenic potential of BIN-67 cells was determined and the tumours formed in a xenograft model was compared to human SCCOHT. DNA sequencing, spectral karyotyping and high density SNP array analysis was performed. The sensitivity of the BIN-67 cells to standard chemotherapeutic agents and to vesicular stomatitis virus (VSV) and the JX-594 vaccinia virus was tested. Results BIN-67 cells were capable of forming spheroids in hanging drop cultures. When xenografted into immunodeficient mice, BIN-67 cells developed into tumours that reflected the hypercalcemia and histology of human SCCOHT, notably intense expression of WT-1 and vimentin, and lack of expression of inhibin. Somatic mutations in TP53 and the most common activating mutations in KRAS and BRAF were not found in BIN-67 cells by DNA sequencing. Spectral karyotyping revealed a largely normal diploid karyotype (in greater than 95% of cells) with a visibly shorter chromosome 20 contig. High density SNP array analysis also revealed few genomic anomalies in BIN-67 cells, which included loss of heterozygosity of an estimated 16.7 Mb interval on chromosome 20. SNP array analyses of four SCCOHT samples also indicated a low frequency of genomic anomalies in the majority of cases. Although resistant to platinum chemotherapeutic drugs, BIN-67 cell viability in vitro was reduced by >75% after infection with oncolytic viruses. Conclusions These results show that SCCOHT differs from high-grade serous carcinomas by exhibiting few chromosomal anomalies and lacking TP53

  4. Unclassified renal cell carcinoma: a clinicopathological, comparative genomic hybridization, and whole-genome exon sequencing study

    PubMed Central

    Hu, Zhen-Yan; Pang, Li-Juan; Qi, Yan; Kang, Xue-Ling; Hu, Jian-Ming; Wang, Lianghai; Liu, Kun-Peng; Ren, Yuan; Cui, Mei; Song, Li-Li; Li, Hong-An; Zou, Hong; Li, Feng

    2014-01-01

    Unclassified renal cell carcinoma (URCC) is a rare variant of RCC, accounting for only 3-5% of all cases. Studies on the molecular genetics of URCC are limited, and hence, we report on 2 cases of URCC analyzed using comparative genome hybridization (CGH) and the genome-wide human exon GeneChip technique to identify the genomic alterations of URCC. Both URCC patients (mean age, 72 years) presented at an advanced stage and died within 30 months post-surgery. Histologically, the URCCs were composed of undifferentiated, multinucleated, giant cells with eosinophilic cytoplasm. Immunostaining revealed that both URCC cases had strong p53 protein expression and partial expression of cluster of differentiation-10 and cytokeratin. The CGH profiles showed chromosomal imbalances in both URCC cases: gains were observed in chromosomes 1p11-12, 1q12-13, 2q20-23, 3q22-23, 8p12, and 16q11-15, whereas losses were detected on chromosomes 1q22-23, 3p12-22, 5p30-ter, 6p, 11q, 16q18-22, 17p12-14, and 20p. Compared with 18 normal renal tissues, 40 mutated genes were detected in the URCC tissues, including 32 missense and 8 silent mutations. Functional enrichment analysis revealed that the missense mutation genes were involved in 11 different biological processes and pathways, including cell cycle regulation, lipid localization and transport, neuropeptide signaling, organic ether metabolism, and ATP-binding cassette transporter signaling. Our findings indicate that URCC may be a highly aggressive cancer, and the genetic alterations identified herein may provide clues regarding the tumorigenesis of URCC and serve as a basis for the development of targeted therapies against URCC in the future. PMID:25120763

  5. Challenges and Triumphs to Genomics-Based Natural Product Discovery

    PubMed Central

    Jensen, Paul R.; Chavarria, Krystle L.; Fenical, William; Moore, Bradley S.; Ziemert, Nadine

    2013-01-01

    Genome sequencing is rapidly changing the field of natural products research by providing opportunities to assess the biosynthetic potential of strains prior to chemical analysis or biological testing. Ready access to sequence data is driving the development of new bioinformatic tools and methods to identify the products of silent or cryptic pathways. While genome mining has fast become a useful approach to natural product discovery, it has also become clear that identifying pathways of interest is much easier than finding the associated products. This has led to bottlenecks in the discovery process that must be overcome for the potential of genomics-based natural product discovery to be fully realized. In this perspective, we address some of these challenges in the context of our work with the marine actinomycete genus Salinispora, which is proving to be a useful model with which to apply genome mining as an approach to natural product discovery. PMID:24104399

  6. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    PubMed

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.

  7. Sequencing Single Cell Microbial Genomes with Microfluidic Amplifications Tools (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    ScienceCinema

    Quake, Steve [University of Stanford

    2016-07-12

    Stanford University's Steve Quake on "Sequencing Single Cell Microbial Genomes with Microfluidic Amplification Tools" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

  8. Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes

    PubMed Central

    2010-01-01

    Background Geobacter species grow by transferring electrons out of the cell - either to Fe(III)-oxides or to man-made substances like energy-harvesting electrodes. Study of Geobacter sulfurreducens has shown that TCA cycle enzymes, inner-membrane respiratory enzymes, and periplasmic and outer-membrane cytochromes are required. Here we present comparative analysis of six Geobacter genomes, including species from the clade that predominates in the subsurface. Conservation of proteins across the genomes was determined to better understand the evolution of Geobacter species and to create a metabolic model applicable to subsurface environments. Results The results showed that enzymes for acetate transport and oxidation, and for proton transport across the inner membrane were well conserved. An NADH dehydrogenase, the ATP synthase, and several TCA cycle enzymes were among the best conserved in the genomes. However, most of the cytochromes required for Fe(III)-reduction were not, including many of the outer-membrane cytochromes. While conservation of cytochromes was poor, an abundance and diversity of cytochromes were found in every genome, with duplications apparent in several species. Conclusions These results indicate there is a common pathway for acetate oxidation and energy generation across the family and in the last common ancestor. They also suggest that while cytochromes are important for extracellular electron transport, the path of electrons across the periplasm and outer membrane is variable. This combination of abundant cytochromes with weak sequence conservation suggests they may not be specific terminal reductases, but rather may be important in their heme-bearing capacity, as sinks for electrons between the inner-membrane electron transport chain and the extracellular acceptor. PMID:20078895

  9. Evidence-based gene predictions in plant genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Automated evidence-based gene building is a rapid and cost-effective way to provide reliable gene annotations on newly sequenced genomes. One of the limitations of evidence-based gene builders, however, is their requirement for gene expression evidence—known proteins, full-length cDNAs, or expressed...

  10. MonarchBase: the monarch butterfly genome database

    PubMed Central

    Zhan, Shuai; Reppert, Steven M.

    2013-01-01

    The monarch butterfly (Danaus plexippus) is emerging as a model organism to study the mechanisms of circadian clocks and animal navigation, and the genetic underpinnings of long-distance migration. The initial assembly of the monarch genome was released in 2011, and the biological interpretation of the genome focused on the butterfly’s migration biology. To make the extensive data associated with the genome accessible to the general biological and lepidopteran communities, we established MonarchBase (available at http://monarchbase.umassmed.edu). The database is an open-access, web-available portal that integrates all available data associated with the monarch butterfly genome. Moreover, MonarchBase provides access to an updated version of genome assembly (v3) upon which all data integration is based. These include genes with systematic annotation, as well as other molecular resources, such as brain expressed sequence tags, migration expression profiles and microRNAs. MonarchBase utilizes a variety of retrieving methods to access data conveniently and for integrating biological interpretations. PMID:23143105

  11. MonarchBase: the monarch butterfly genome database.

    PubMed

    Zhan, Shuai; Reppert, Steven M

    2013-01-01

    The monarch butterfly (Danaus plexippus) is emerging as a model organism to study the mechanisms of circadian clocks and animal navigation, and the genetic underpinnings of long-distance migration. The initial assembly of the monarch genome was released in 2011, and the biological interpretation of the genome focused on the butterfly's migration biology. To make the extensive data associated with the genome accessible to the general biological and lepidopteran communities, we established MonarchBase (available at http://monarchbase.umassmed.edu). The database is an open-access, web-available portal that integrates all available data associated with the monarch butterfly genome. Moreover, MonarchBase provides access to an updated version of genome assembly (v3) upon which all data integration is based. These include genes with systematic annotation, as well as other molecular resources, such as brain expressed sequence tags, migration expression profiles and microRNAs. MonarchBase utilizes a variety of retrieving methods to access data conveniently and for integrating biological interpretations.

  12. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    PubMed Central

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna; Shoaie, Saeed; Kampf, Caroline; Uhlen, Mathias; Nielsen, Jens

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted 85 antimetabolites that can inhibit growth of, or even kill, any of the cell lines, while at the same time not being toxic for 83 different healthy human cell types. 60 of these antimetabolites were found to inhibit growth in all cell lines. Finally, we experimentally validated one of the predicted antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies. PMID:25640694

  13. Insights from a chimpanzee adipose stromal cell population: opportunities for adult stem cells to expand primate functional genomics.

    PubMed

    Pfefferle, Lisa W; Wray, Gregory A

    2013-01-01

    Comparisons between humans and chimpanzees are essential for understanding traits unique to each species. However, linking important phenotypic differences to underlying molecular changes is often challenging. The ability to generate, differentiate, and profile adult stem cells provides a powerful but underutilized opportunity to investigate the molecular basis for trait differences between species within specific cell types and in a controlled environment. Here, we characterize adipose stromal cells (ASCs) from Clint, the chimpanzee whose genome was first sequenced. Using imaging and RNA-Seq, we compare the chimpanzee ASCs with three comparable human cell lines. Consistent with previous studies on ASCs in humans, the chimpanzee cells have fibroblast-like morphology and express genes encoding components of the extracellular matrix at high levels. Differentially expressed genes are enriched for distinct functional classes between species: immunity and protein processing are higher in chimpanzees, whereas cell cycle and DNA processing are higher in humans. Although hesitant to draw definitive conclusions from these data given the limited sample size, we wish to stress the opportunities that adult stem cells offer for studying primate evolution. In particular, adult stem cells provide a powerful means to investigate the profound disease susceptibilities unique to humans and a promising tool for conservation efforts with nonhuman primates. By allowing for experimental perturbations in relevant cell types, adult stem cells promise to complement classic comparative primate genomics based on in vivo sampling.

  14. CRISPR mediated somatic cell genome engineering in the chicken.

    PubMed

    Véron, Nadège; Qu, Zhengdong; Kipen, Phoebe A S; Hirst, Claire E; Marcelle, Christophe

    2015-11-01

    Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model.

  15. Genomic portfolio of Merkel cell carcinoma as determined by comprehensive genomic profiling: implications for targeted therapeutics.

    PubMed

    Cohen, Philip R; Tomson, Brett N; Elkin, Sheryl K; Marchlik, Erica; Carter, Jennifer L; Kurzrock, Razelle

    2016-04-26

    Merkel cell carcinoma is an ultra-rare cutaneous neuroendocrine cancer for which approved treatment options are lacking. To better understand potential actionability, the genomic landscape of Merkel cell cancers was assessed. The molecular aberrations in 17 patients with Merkel cell carcinoma were, on physician request, tested in a Clinical Laboratory Improvement Amendments (CLIA) laboratory (Foundation Medicine, Cambridge, MA) using next-generation sequencing (182 or 236 genes) and analyzed by N-of-One, Inc. (Lexington, MA). There were 30 genes harboring aberrations and 60 distinct molecular alterations identified in this patient population. The most common abnormalities involved the TP53 gene (12/17 [71% of patients]) and the cell cycle pathway (CDKN2A/B, CDKN2C or RB1) (12/17 [71%]). Abnormalities also were observed in the PI3K/AKT/mTOR pathway (AKT2, FBXW7, NF1, PIK3CA, PIK3R1, PTEN or RICTOR) (9/17 [53%]) and DNA repair genes (ATM, BAP1, BRCA1/2, CHEK2, FANCA or MLH1) (5/17 [29%]). Possible cognate targeted therapies, including FDA-approved drugs, could be identified in most of the patients (16/17 [94%]). In summary, Merkel cell carcinomas were characterized by multiple distinct aberrations that were unique in the majority of analyzed cases. Most patients had theoretically actionable alterations. These results provide a framework for investigating tailored combinations of matched therapies in Merkel cell carcinoma patients.

  16. Stem-like cancer cells are inducible by increasing genomic instability in cancer cells.

    PubMed

    Liang, Yi; Zhong, Zhendong; Huang, Yijun; Deng, Wen; Cao, Junxia; Tsao, George; Liu, Quentin; Pei, Duanqing; Kang, Tiebang; Zeng, Yi-Xin

    2010-02-12

    The existence of cancer stem cells (CSCs) or stem-like cancer cells (SLCCs) is regarded as the cause of tumor formation and recurrence. However, the origin of such cells remains controversial with two competing hypotheses: CSCs are either transformed from tissue adult stem cells or dedifferentiated from transformed progenitor cells. Compelling evidence has determined the chromosomal aneuploidy to be one of the hallmarks of cancer cells, indicating genome instability plays an important role in tumorigenesis, for which CSCs are believed to be the initiator. To gain direct evidence that genomic instability is involved in the induction of SLCCs, we utilized multiple approaches to enhance genomic instability and monitored the percentage of SLCC in cultured cancer cells. Using side population (SP) cells as a marker for SLCC in human nasopharyngeal carcinoma (NPC) and CD133 for human neuroblastoma cells, we found that DNA damage inducers, UV and mitomycin C were capable of increasing SP cells in NPC CNE-2 and neuroblastoma SKN-SH cells. Likewise, either overexpression of a key regulator of cell cycle, Mad2, or knock down of Aurora B, an important kinase in mitosis, or Cdh1, a key E3 ligase in cell cycle, resulted in a significant increase of SP cells in CNE-2. More interestingly, enrichment of SP cells was observed in recurrent tumor tissues as compared with the primary tumor in the same NPC patients. Our study thus suggested that, beside transformation of tissue stem cells leading to CSC generation, genomic instability could be another potential mechanism resulting in SLCC formation, especially at tumor recurrence stage.

  17. Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells

    PubMed Central

    Ruiz, Sergio; Lopez-Contreras, Andres J.; Gabut, Mathieu; Marion, Rosa M.; Gutierrez-Martinez, Paula; Bua, Sabela; Ramirez, Oscar; Olalde, Iñigo; Rodrigo-Perez, Sara; Li, Han; Marques-Bonet, Tomas; Serrano, Manuel; Blasco, Maria A.; Batada, Nizar N.; Fernandez-Capetillo, Oscar

    2015-01-01

    The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC. PMID:26292731

  18. Emerging paradigms in genomics-based crop improvement.

    PubMed

    Bohra, Abhishek

    2013-11-17

    Next generation sequencing platforms and high-throughput genotyping assays have remarkably expedited the pace of development of genomic tools and resources for several crops. Complementing the technological developments, conceptual shifts have also been witnessed in designing experimental populations. Availability of second generation mapping populations encompassing multiple alleles, multiple traits, and extensive recombination events is radically changing the phenomenon of classical QTL mapping. Additionally, the rising molecular breeding approaches like marker assisted recurrent selection (MARS) that are able to harness several QTLs are of particular importance in obtaining a "designed" genotype carrying the most desirable combinations of favourable alleles. Furthermore, rapid generation of genome-wide marker data coupled with easy access to precise and accurate phenotypic screens enable large-scale exploitation of LD not only to discover novel QTLs via whole genome association scans but also to practise genomic estimated breeding value (GEBV)-based selection of genotypes. Given refinements being experienced in analytical methods and software tools, the multiparent populations will be the resource of choice to undertake genome wide association studies (GWAS), multiparent MARS, and genomic selection (GS). With this, it is envisioned that these high-throughput and high-power molecular breeding methods would greatly assist in exploiting the enormous potential underlying breeding by design approach to facilitate accelerated crop improvement.

  19. Genome-wide examination of myoblast cell cycle withdrawal duringdifferentiation

    SciTech Connect

    Shen, Xun; Collier, John Michael; Hlaing, Myint; Zhang, Leanne; Delshad, Elizabeth H.; Bristow, James; Bernstein, Harold S.

    2002-12-02

    Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40 percent fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly

  20. Genomics of Drug Sensitivity in Cancer (GDSC): a resource for therapeutic biomarker discovery in cancer cells.

    PubMed

    Yang, Wanjuan; Soares, Jorge; Greninger, Patricia; Edelman, Elena J; Lightfoot, Howard; Forbes, Simon; Bindal, Nidhi; Beare, Dave; Smith, James A; Thompson, I Richard; Ramaswamy, Sridhar; Futreal, P Andrew; Haber, Daniel A; Stratton, Michael R; Benes, Cyril; McDermott, Ultan; Garnett, Mathew J

    2013-01-01

    Alterations in cancer genomes strongly influence clinical responses to treatment and in many instances are potent biomarkers for response to drugs. The Genomics of Drug Sensitivity in Cancer (GDSC) database (www.cancerRxgene.org) is the largest public resource for information on drug sensitivity in cancer cells and molecular markers of drug response. Data are freely available without restriction. GDSC currently contains drug sensitivity data for almost 75 000 experiments, describing response to 138 anticancer drugs across almost 700 cancer cell lines. To identify molecular markers of drug response, cell line drug sensitivity data are integrated with large genomic datasets obtained from the Catalogue of Somatic Mutations in Cancer database, including information on somatic mutations in cancer genes, gene amplification and deletion, tissue type and transcriptional data. Analysis of GDSC data is through a web portal focused on identifying molecular biomarkers of drug sensitivity based on queries of specific anticancer drugs or cancer genes. Graphical representations of the data are used throughout with links to related resources and all datasets are fully downloadable. GDSC provides a unique resource incorporating large drug sensitivity and genomic datasets to facilitate the discovery of new therapeutic biomarkers for cancer therapies.

  1. Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time

    PubMed Central

    Feichtinger, Julia; Hernández, Inmaculada; Fischer, Christoph; Hanscho, Michael; Auer, Norbert; Hackl, Matthias; Jadhav, Vaibhav; Baumann, Martina; Krempl, Peter M.; Schmidl, Christian; Farlik, Matthias; Schuster, Michael; Merkel, Angelika; Sommer, Andreas; Heath, Simon; Rico, Daniel; Bock, Christoph; Thallinger, Gerhard G.

    2016-01-01

    ABSTRACT The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards phenotype evolution, comprehensive genome and epigenome data are presented for six related CHO cell lines, both in response to perturbations (different culture conditions and media as well as selection of a specific phenotype with increased transient productivity) and in steady state (prolonged time in culture under constant conditions). Clear transitions were observed in DNA‐methylation patterns upon each perturbation, while few changes occurred over time under constant conditions. Only minor DNA‐methylation changes were observed between exponential and stationary growth phase; however, throughout a batch culture the histone modification pattern underwent continuous adaptation. Variation in genome sequence between the six cell lines on the level of SNPs, InDels, and structural variants is high, both upon perturbation and under constant conditions over time. The here presented comprehensive resource may open the door to improved control and manipulation of gene expression during industrial bioprocesses based on epigenetic mechanisms. Biotechnol. Bioeng. 2016;113: 2241–2253. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:27072894

  2. Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure.

    PubMed

    Yan, Qiang; Xu, Kun; Xing, Jiani; Zhang, Tingting; Wang, Xin; Wei, Zehui; Ren, Chonghua; Liu, Zhongtian; Shao, Simin; Zhang, Zhiying

    2016-12-12

    The clustered regularly interspaced short palindromic repeats (CRISPR) system has recently been developed into a powerful genome-editing technology, as it requires only two key components (Cas9 protein and sgRNA) to function and further enables multiplex genome targeting and homology-directed repair (HDR) based precise genome editing in a wide variety of organisms. Here, we report a novel and interesting strategy by using the Drosha-mediated sgRNA-shRNA structure to direct Cas9 for multiplex genome targeting and precise genome editing. For multiplex genome targeting assay, we achieved more than 9% simultaneous mutant efficiency for 3 genomic loci among the puromycin-selected cell clones. By introducing the shRNA against DNA ligase IV gene (LIG4) into the sgRNA-shRNA construct, the HDR-based precise genome editing efficiency was improved as more than 2-fold. Our works provide a useful tool for multiplex and precise genome modifying in mammalian cells.

  3. Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure

    PubMed Central

    Yan, Qiang; Xu, Kun; Xing, Jiani; Zhang, Tingting; Wang, Xin; Wei, Zehui; Ren, Chonghua; Liu, Zhongtian; Shao, Simin; Zhang, Zhiying

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system has recently been developed into a powerful genome-editing technology, as it requires only two key components (Cas9 protein and sgRNA) to function and further enables multiplex genome targeting and homology-directed repair (HDR) based precise genome editing in a wide variety of organisms. Here, we report a novel and interesting strategy by using the Drosha-mediated sgRNA-shRNA structure to direct Cas9 for multiplex genome targeting and precise genome editing. For multiplex genome targeting assay, we achieved more than 9% simultaneous mutant efficiency for 3 genomic loci among the puromycin-selected cell clones. By introducing the shRNA against DNA ligase IV gene (LIG4) into the sgRNA-shRNA construct, the HDR-based precise genome editing efficiency was improved as more than 2-fold. Our works provide a useful tool for multiplex and precise genome modifying in mammalian cells. PMID:27941919

  4. Protein and genome evolution in Mammalian cells for biotechnology applications.

    PubMed

    Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J

    2009-06-01

    Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.

  5. Duplication of the genome in normal and cancer cell cycles.

    PubMed

    Bandura, Jennifer L; Calvi, Brian R

    2002-01-01

    It is critical to discover the mechanisms of normal cell cycle regulation if we are to fully understand what goes awry in cancer cells. The normal eukaryotic cell tightly regulates the activity of origins of DNA replication so that the genome is duplicated exactly once per cell cycle. Over the last ten years much has been learned concerning the cell cycle regulation of origin activity. It is now clear that the proteins and cell cycle mechanisms that control origin activity are largely conserved from yeast to humans. Despite this conservation, the composition of origins of DNA replication in higher eukaryotes remains ill defined. A DNA consensus for predicting origins has yet to emerge, and it is of some debate whether primary DNA sequence determines where replication initiates. In this review we outline what is known about origin structure and the mechanism of once per cell cycle DNA replication with an emphasis on recent advances in mammalian cells. We discuss the possible relevance of these regulatory pathways for cancer biology and therapy.

  6. Each cell counts: Hematopoiesis and immunity research in the era of single cell genomics.

    PubMed

    Jaitin, Diego Adhemar; Keren-Shaul, Hadas; Elefant, Naama; Amit, Ido

    2015-02-01

    Hematopoiesis and immunity are mediated through complex interactions between multiple cell types and states. This complexity is currently addressed following a reductionist approach of characterizing cell types by a small number of cell surface molecular features and gross functions. While the introduction of global transcriptional profiling technologies enabled a more comprehensive view, heterogeneity within sampled populations remained unaddressed, obscuring the true picture of hematopoiesis and immune system function. A critical mass of technological advances in molecular biology and genomics has enabled genome-wide measurements of single cells - the fundamental unit of immunity. These new advances are expected to boost detection of less frequent cell types and fuzzy intermediate cell states, greatly expanding the resolution of current available classifications. This new era of single-cell genomics in immunology research holds great promise for further understanding of the mechanisms and circuits regulating hematopoiesis and immunity in both health and disease. In the near future, the accuracy of single-cell genomics will ultimately enable precise diagnostics and treatment of multiple hematopoietic and immune related diseases.

  7. Genome-based prediction of testcross values in maize.

    PubMed

    Albrecht, Theresa; Wimmer, Valentin; Auinger, Hans-Jürgen; Erbe, Malena; Knaak, Carsten; Ouzunova, Milena; Simianer, Henner; Schön, Chris-Carolin

    2011-07-01

    This is the first large-scale experimental study on genome-based prediction of testcross values in an advanced cycle breeding population of maize. The study comprised testcross progenies of 1,380 doubled haploid lines of maize derived from 36 crosses and phenotyped for grain yield and grain dry matter content in seven locations. The lines were genotyped with 1,152 single nucleotide polymorphism markers. Pedigree data were available for three generations. We used best linear unbiased prediction and stratified cross-validation to evaluate the performance of prediction models differing in the modeling of relatedness between inbred lines and in the calculation of genome-based coefficients of similarity. The choice of similarity coefficient did not affect prediction accuracies. Models including genomic information yielded significantly higher prediction accuracies than the model based on pedigree information alone. Average prediction accuracies based on genomic data were high even for a complex trait like grain yield (0.72-0.74) when the cross-validation scheme allowed for a high degree of relatedness between the estimation and the test set. When predictions were performed across distantly related families, prediction accuracies decreased significantly (0.47-0.48). Prediction accuracies decreased with decreasing sample size but were still high when the population size was halved (0.67-0.69). The results from this study are encouraging with respect to genome-based prediction of the genetic value of untested lines in advanced cycle breeding populations and the implementation of genomic selection in the breeding process.

  8. Carcinogens induce genome-wide loss of heterozygosity in normal stem cells without persistent chromosomal instability.

    PubMed

    Donahue, Sarah L; Lin, Qing; Cao, Shang; Ruley, H Earl

    2006-08-01

    Widespread losses of heterozygosity (LOH) in human cancer have been thought to result from chromosomal instability caused by mutations affecting DNA repair/genome maintenance. However, the origin of LOH in most tumors is unknown. The present study examined the ability of carcinogenic agents to induce LOH at 53 sites throughout the genome of normal diploid mouse ES cells. Brief exposures to nontoxic levels of methylnitrosourea, diepoxybutane, mitomycin C, hydroxyurea, doxorubicin, and UV light stimulated LOH at all loci at frequencies ranging from 1-8 x 10(-3) per cell (10-123 times higher than in untreated cells). This greatly exceeds the frequencies at which these agents have been reported to induce point mutations and is comparable to the rates of LOH observed in ES cells lacking the gene responsible for Bloom syndrome, an inherited DNA repair defect that results in greatly increased risk of cancer. These results suggest that LOH contributes significantly to the carcinogenicity of a variety of mutagens and raises the possibility that genome-wide LOH observed in some human cancers may reflect prior exposure to genotoxic agents rather than a state of chromosomal instability during the carcinogenic process. Finally, as a practical matter, chemically induced LOH is expected to enhance the recovery of homozygous recessive mutants from phenotype-based genetic screens in mammalian cells.

  9. Carcinogens induce genome-wide loss of heterozygosity in normal stem cells without persistent chromosomal instability

    PubMed Central

    Donahue, Sarah L.; Lin, Qing; Cao, Shang; Ruley, H. Earl

    2006-01-01

    Widespread losses of heterozygosity (LOH) in human cancer have been thought to result from chromosomal instability caused by mutations affecting DNA repair/genome maintenance. However, the origin of LOH in most tumors is unknown. The present study examined the ability of carcinogenic agents to induce LOH at 53 sites throughout the genome of normal diploid mouse ES cells. Brief exposures to nontoxic levels of methylnitrosourea, diepoxybutane, mitomycin C, hydroxyurea, doxorubicin, and UV light stimulated LOH at all loci at frequencies ranging from 1–8 × 10−3 per cell (10–123 times higher than in untreated cells). This greatly exceeds the frequencies at which these agents have been reported to induce point mutations and is comparable to the rates of LOH observed in ES cells lacking the gene responsible for Bloom syndrome, an inherited DNA repair defect that results in greatly increased risk of cancer. These results suggest that LOH contributes significantly to the carcinogenicity of a variety of mutagens and raises the possibility that genome-wide LOH observed in some human cancers may reflect prior exposure to genotoxic agents rather than a state of chromosomal instability during the carcinogenic process. Finally, as a practical matter, chemically induced LOH is expected to enhance the recovery of homozygous recessive mutants from phenotype-based genetic screens in mammalian cells. PMID:16868089

  10. WormBase 2016: expanding to enable helminth genomic research

    PubMed Central

    Howe, Kevin L.; Bolt, Bruce J.; Cain, Scott; Chan, Juancarlos; Chen, Wen J.; Davis, Paul; Done, James; Down, Thomas; Gao, Sibyl; Grove, Christian; Harris, Todd W.; Kishore, Ranjana; Lee, Raymond; Lomax, Jane; Li, Yuling; Muller, Hans-Michael; Nakamura, Cecilia; Nuin, Paulo; Paulini, Michael; Raciti, Daniela; Schindelman, Gary; Stanley, Eleanor; Tuli, Mary Ann; Van Auken, Kimberly; Wang, Daniel; Wang, Xiaodong; Williams, Gary; Wright, Adam; Yook, Karen; Berriman, Matthew; Kersey, Paul; Schedl, Tim; Stein, Lincoln; Sternberg, Paul W.

    2016-01-01

    WormBase (www.wormbase.org) is a central repository for research data on the biology, genetics and genomics of Caenorhabditis elegans and other nematodes. The project has evolved from its original remit to collect and integrate all data for a single species, and now extends to numerous nematodes, ranging from evolutionary comparators of C. elegans to parasitic species that threaten plant, animal and human health. Research activity using C. elegans as a model system is as vibrant as ever, and we have created new tools for community curation in response to the ever-increasing volume and complexity of data. To better allow users to navigate their way through these data, we have made a number of improvements to our main website, including new tools for browsing genomic features and ontology annotations. Finally, we have developed a new portal for parasitic worm genomes. WormBase ParaSite (parasite.wormbase.org) contains all publicly available nematode and platyhelminth annotated genome sequences, and is designed specifically to support helminth genomic research. PMID:26578572

  11. Changing Histopathological Diagnostics by Genome-Based Tumor Classification

    PubMed Central

    Kloth, Michael; Buettner, Reinhard

    2014-01-01

    Traditionally, tumors are classified by histopathological criteria, i.e., based on their specific morphological appearances. Consequently, current therapeutic decisions in oncology are strongly influenced by histology rather than underlying molecular or genomic aberrations. The increase of information on molecular changes however, enabled by the Human Genome Project and the International Cancer Genome Consortium as well as the manifold advances in molecular biology and high-throughput sequencing techniques, inaugurated the integration of genomic information into disease classification. Furthermore, in some cases it became evident that former classifications needed major revision and adaption. Such adaptations are often required by understanding the pathogenesis of a disease from a specific molecular alteration, using this molecular driver for targeted and highly effective therapies. Altogether, reclassifications should lead to higher information content of the underlying diagnoses, reflecting their molecular pathogenesis and resulting in optimized and individual therapeutic decisions. The objective of this article is to summarize some particularly important examples of genome-based classification approaches and associated therapeutic concepts. In addition to reviewing disease specific markers, we focus on potentially therapeutic or predictive markers and the relevance of molecular diagnostics in disease monitoring. PMID:24879454

  12. Phylogenetic analyses of phylum Actinobacteria based on whole genome sequences.

    PubMed

    Verma, Mansi; Lal, Devi; Kaur, Jaspreet; Saxena, Anjali; Kaur, Jasvinder; Anand, Shailly; Lal, Rup

    2013-09-01

    Actinobacteria constitute one of the largest and ancient taxonomic phylum within the domain bacteria and are well known for their secondary metabolites. Considerable variation in the metabolic properties, genome size and GC content of the members of this phylum has been observed. Therefore, the placement of new or existing species based on 16S rRNA gene sometimes becomes problematic due to the low congruence level. In the present study, phylogeny of ninety actinobacterial genomes was reconstructed using single gene and whole genome based data. Where alignment-free phylogenetic method was found to be more robust, the concatenation of 94 proteins improved the resolution which all single gene based phylogenies failed to resolve. The comprehensive analysis of 94 conserved proteins resulted in a total of 42,447 informative sites, which is so far the largest meta-alignment obtained for this phylum. But the ultimate resolved phylogeny was obtained by generating a consensus tree by combining the information from single gene and genome based phylogenies. The present investigation clearly revealed that the consensus approach is a useful tool for phylogenetic inference and the taxonomic affiliations must be based on this approach. The consensus approach suggested that there is a need for taxonomic amendments of the orders Frankiales and Micrococcales.

  13. Genomic instability, driver genes and cell selection: Projections from cancer to stem cells.

    PubMed

    Ben-David, Uri

    2015-04-01

    Cancer cells and stem cells share many traits, including a tendency towards genomic instability. Human cancers exhibit tumor-specific genomic aberrations, which often affect their malignancy and drug response. During their culture propagation, human pluripotent stem cells (hPSCs) also acquire characteristic genomic aberrations, which may have significant impact on their molecular and cellular phenotypes. These aberrations vary in size from single nucleotide alterations to copy number alterations to whole chromosome gains. A prominent challenge in both cancer and stem cell research is to identify "driver aberrations" that confer a selection advantage, and "driver genes" that underlie the recurrence of these aberrations. Following principles that are already well-established in cancer research, candidate driver genes have also been suggested in hPSCs. Experimental validation of the functional role of such candidates can uncover whether these are bona fide driver genes. The identification of driver genes may bring us closer to a mechanistic understanding of the genomic instability of stem cells. Guided by terminologies and methodologies commonly applied in cancer research, such understanding may have important ramifications for both stem cell and cancer biology. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

  14. Sleeping Beauty Transposon Mutagenesis of the Rat Genome in Spermatogonial Stem Cells

    PubMed Central

    Ivics, Zoltán; Izsvák, Zsuzsanna; Chapman, Karen M.; Hamra, F. Kent

    2011-01-01

    Since several aspects of physiology in rats has evolved to be more similar to humans than that of mice, it is highly desirable to link the rat into the process of annotating the human genome with function. However, the lack of technology for generating defined mutants in the rat genome has hindered the identification of causative relationships between genes and disease phenotypes. As an important step towards this goal, an approach of establishing transposon-mediated insertional mutagenesis in rat spermatogonial stem cells was recently developed. Transposons can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of transposons can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest such as a mutagenic gene trap cassette cloned between the inverted repeat sequences of a transposon-based vector can be utilized for stable genomic insertion in a regulated and highly efficient manner. Gene trap transposons integrate into the genome in a random fashion, and those mutagenic insertions that occurred in expressed genes can be selected in vitro based on activation of a reporter. Selected monoclonal as well as polyclonal libraries of gene trap clones are transplanted into the testes of recipient/founder male rats allowing passage of the mutation through the germline to F1 progeny after only a single cross with wild-type females. This paradigm enables a powerful methodological pipeline for forward genetic screens for functional gene annotation in the rat, as well as other vertebrate models. This article provides a detailed description on how to culturerat spermatogonial stem cell lines, their transfection with transposon plasmids, selection of gene trap insertions with antibiotics, transplantation of genetically modified stem cells and genotyping of knockout animals. PMID:21193047

  15. Molecular Mechanisms Underlying Genomic Instability in Brca-Deficient Cells

    DTIC Science & Technology

    2014-11-01

    homology-directed repair, which is counteracted by 53BP1, BRCA2 functions later by promoting RAD51 filament formation. We have generated BRCA2/PTIP-doubly...Interestingly, breaks in the vicinity of the GIMAP hot spot were detectable spontaneously in XRCC2/ cells (Figures 3C and 3D ; Table S3), which is...and a nonrandom 3D organization of the genome (Chiarle et al., 2011; Hakim et al., 2012; Klein et al., 2011; Zhang et al., 2012b). To date, replication

  16. Malignant glioma: lessons from genomics, mouse models, and stem cells.

    PubMed

    Chen, Jian; McKay, Renée M; Parada, Luis F

    2012-03-30

    Eighty percent of malignant tumors that develop in the central nervous system are malignant gliomas, which are essentially incurable. Here, we discuss how recent sequencing studies are identifying unexpected drivers of gliomagenesis, including mutations in isocitrate dehydrogenase 1 and the NF-κB pathway, and how genome-wide analyses are reshaping the classification schemes for tumors and enhancing prognostic value of molecular markers. We discuss the controversies surrounding glioma stem cells and explore how the integration of new molecular data allows for the generation of more informative animal models to advance our knowledge of glioma's origin, progression, and treatment.

  17. Advances in genome-wide protein expression using the wheat germ cell-free system.

    PubMed

    Endo, Yaeta; Sawasaki, Tatsuya

    2005-01-01

    In the current post-genomic era, cell-free translation platforms are gaining importance in structural as well as functional genomics. They are based on extracts prepared from Escherichia coli cells, wheat germ, or rabbit reticulocytes, and when programmed with any mRNA in the presence of energy sources and amino acids, can synthesize the respective protein in vitro. Among the cell-free systems, the wheat germ-based translation system is of special interest due to its eukaryotic nature and robustness. This chapter outlines the existing protein production platforms and their limitations, and describes the basic concept of the wheat germ-based cell-free system. It also demonstrates how the conventional wheat germ system can be improved by eliminating endogenous inhibitors, by using an expression vector specially designed for this system and polymerase chain reaction-directed protein synthesis directly from cDNAs in a bi-layer translation system. Finally, a robotic procedure for translation based on the wheat germ extract and bi-layer cell-free translation is described.

  18. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.

    PubMed

    Miyaoka, Yuichiro; Berman, Jennifer R; Cooper, Samantha B; Mayerl, Steven J; Chan, Amanda H; Zhang, Bin; Karlin-Neumann, George A; Conklin, Bruce R

    2016-03-31

    Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

  19. GI-SVM: A sensitive method for predicting genomic islands based on unannotated sequence of a single genome.

    PubMed

    Lu, Bingxin; Leong, Hon Wai

    2016-02-01

    Genomic islands (GIs) are clusters of functionally related genes acquired by lateral genetic transfer (LGT), and they are present in many bacterial genomes. GIs are extremely important for bacterial research, because they not only promote genome evolution but also contain genes that enhance adaption and enable antibiotic resistance. Many methods have been proposed to predict GI. But most of them rely on either annotations or comparisons with other closely related genomes. Hence these methods cannot be easily applied to new genomes. As the number of newly sequenced bacterial genomes rapidly increases, there is a need for methods to detect GI based solely on sequences of a single genome. In this paper, we propose a novel method, GI-SVM, to predict GIs given only the unannotated genome sequence. GI-SVM is based on one-class support vector machine (SVM), utilizing composition bias in terms of k-mer content. From our evaluations on three real genomes, GI-SVM can achieve higher recall compared with current methods, without much loss of precision. Besides, GI-SVM allows flexible parameter tuning to get optimal results for each genome. In short, GI-SVM provides a more sensitive method for researchers interested in a first-pass detection of GI in newly sequenced genomes.

  20. Single-Cell-Genomics-Facilitated Read Binning of Candidate Phylum EM19 Genomes from Geothermal Spring Metagenomes.

    PubMed

    Becraft, Eric D; Dodsworth, Jeremy A; Murugapiran, Senthil K; Ohlsson, J Ingemar; Briggs, Brandon R; Kanbar, Jad; De Vlaminck, Iwijn; Quake, Stephen R; Dong, Hailiang; Hedlund, Brian P; Swingley, Wesley D

    2015-12-04

    The vast majority of microbial life remains uncatalogued due to the inability to cultivate these organisms in the laboratory. This "microbial dark matter" represents a substantial portion of the tree of life and of the populations that contribute to chemical cycling in many ecosystems. In this work, we leveraged an existing single-cell genomic data set representing the candidate bacterial phylum "Calescamantes" (EM19) to calibrate machine learning algorithms and define metagenomic bins directly from pyrosequencing reads derived from Great Boiling Spring in the U.S. Great Basin. Compared to other assembly-based methods, taxonomic binning with a read-based machine learning approach yielded final assemblies with the highest predicted genome completeness of any method tested. Read-first binning subsequently was used to extract Calescamantes bins from all metagenomes with abundant Calescamantes populations, including metagenomes from Octopus Spring and Bison Pool in Yellowstone National Park and Gongxiaoshe Spring in Yunnan Province, China. Metabolic reconstruction suggests that Calescamantes are heterotrophic, facultative anaerobes, which can utilize oxidized nitrogen sources as terminal electron acceptors for respiration in the absence of oxygen and use proteins as their primary carbon source. Despite their phylogenetic divergence, the geographically separate Calescamantes populations were highly similar in their predicted metabolic capabilities and core gene content, respiring O2, or oxidized nitrogen species for energy conservation in distant but chemically similar hot springs.

  1. Single-Cell-Genomics-Facilitated Read Binning of Candidate Phylum EM19 Genomes from Geothermal Spring Metagenomes

    PubMed Central

    Becraft, Eric D.; Dodsworth, Jeremy A.; Murugapiran, Senthil K.; Ohlsson, J. Ingemar; Briggs, Brandon R.; Kanbar, Jad; De Vlaminck, Iwijn; Quake, Stephen R.; Dong, Hailiang; Hedlund, Brian P.

    2015-01-01

    The vast majority of microbial life remains uncatalogued due to the inability to cultivate these organisms in the laboratory. This “microbial dark matter” represents a substantial portion of the tree of life and of the populations that contribute to chemical cycling in many ecosystems. In this work, we leveraged an existing single-cell genomic data set representing the candidate bacterial phylum “Calescamantes” (EM19) to calibrate machine learning algorithms and define metagenomic bins directly from pyrosequencing reads derived from Great Boiling Spring in the U.S. Great Basin. Compared to other assembly-based methods, taxonomic binning with a read-based machine learning approach yielded final assemblies with the highest predicted genome completeness of any method tested. Read-first binning subsequently was used to extract Calescamantes bins from all metagenomes with abundant Calescamantes populations, including metagenomes from Octopus Spring and Bison Pool in Yellowstone National Park and Gongxiaoshe Spring in Yunnan Province, China. Metabolic reconstruction suggests that Calescamantes are heterotrophic, facultative anaerobes, which can utilize oxidized nitrogen sources as terminal electron acceptors for respiration in the absence of oxygen and use proteins as their primary carbon source. Despite their phylogenetic divergence, the geographically separate Calescamantes populations were highly similar in their predicted metabolic capabilities and core gene content, respiring O2, or oxidized nitrogen species for energy conservation in distant but chemically similar hot springs. PMID:26637598

  2. Whole-genome duplication increases tumor cell sensitivity to MPS1 inhibition

    PubMed Central

    Jemaà, Mohamed; Manic, Gwenola; Lledo, Gwendaline; Lissa, Delphine; Reynes, Christelle; Morin, Nathalie; Chibon, Frédéric; Sistigu, Antonella; Castedo, Maria; Vitale, Ilio; Kroemer, Guido; Abrieu, Ariane

    2016-01-01

    Several lines of evidence indicate that whole-genome duplication resulting in tetraploidy facilitates carcinogenesis by providing an intermediate and metastable state more prone to generate oncogenic aneuploidy. Here, we report a novel strategy to preferentially kill tetraploid cells based on the abrogation of the spindle assembly checkpoint (SAC) via the targeting of TTK protein kinase (better known as monopolar spindle 1, MPS1). The pharmacological inhibition as well as the knockdown of MPS1 kills more efficiently tetraploid cells than their diploid counterparts. By using time-lapse videomicroscopy, we show that tetraploid cells do not survive the aborted mitosis due to SAC abrogation upon MPS1 depletion. On the contrary diploid cells are able to survive up to at least two more cell cycles upon the same treatment. This effect might reflect the enhanced difficulty of cells with whole-genome doubling to tolerate a further increase in ploidy and/or an elevated level of chromosome instability in the absence of SAC functions. We further show that MPS1-inhibited tetraploid cells promote mitotic catastrophe executed by the intrinsic pathway of apoptosis, as indicated by the loss of mitochondrial potential, the release of the pro-apoptotic cytochrome c from mitochondria, and the activation of caspases. Altogether, our results suggest that MPS1 inhibition could be used as a therapeutic strategy for targeting tetraploid cancer cells. PMID:26637805

  3. Comprehensive red blood cell and platelet antigen prediction from whole genome sequencing: proof of principle

    PubMed Central

    Westhoff, Connie M.; Uy, Jon Michael; Aguad, Maria; Smeland‐Wagman, Robin; Kaufman, Richard M.; Rehm, Heidi L.; Green, Robert C.; Silberstein, Leslie E.

    2015-01-01

    BACKGROUND There are 346 serologically defined red blood cell (RBC) antigens and 33 serologically defined platelet (PLT) antigens, most of which have known genetic changes in 45 RBC or six PLT genes that correlate with antigen expression. Polymorphic sites associated with antigen expression in the primary literature and reference databases are annotated according to nucleotide positions in cDNA. This makes antigen prediction from next‐generation sequencing data challenging, since it uses genomic coordinates. STUDY DESIGN AND METHODS The conventional cDNA reference sequences for all known RBC and PLT genes that correlate with antigen expression were aligned to the human reference genome. The alignments allowed conversion of conventional cDNA nucleotide positions to the corresponding genomic coordinates. RBC and PLT antigen prediction was then performed using the human reference genome and whole genome sequencing (WGS) data with serologic confirmation. RESULTS Some major differences and alignment issues were found when attempting to convert the conventional cDNA to human reference genome sequences for the following genes: ABO, A4GALT, RHD, RHCE, FUT3, ACKR1 (previously DARC), ACHE, FUT2, CR1, GCNT2, and RHAG. However, it was possible to create usable alignments, which facilitated the prediction of all RBC and PLT antigens with a known molecular basis from WGS data. Traditional serologic typing for 18 RBC antigens were in agreement with the WGS‐based antigen predictions, providing proof of principle for this approach. CONCLUSION Detailed mapping of conventional cDNA annotated RBC and PLT alleles can enable accurate prediction of RBC and PLT antigens from whole genomic sequencing data. PMID:26634332

  4. Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CRISPR/Cas9 has been recently demonstrated as an effective and popular genome editing tool for modifying genomes of human, animals, microorganisms, and plants. Success of such genome editing is highly dependent on the availability of suitable target sites in the genomes to be edited. Many specific t...

  5. INTEGRATED GENOME-BASED STUDIES OF SHEWANELLA ECOPHYSIOLOGY

    SciTech Connect

    TIEDJE, JAMES M; KONSTANTINIDIS, KOSTAS; WORDEN, MARK

    2014-01-08

    The aim of the work reported is to study Shewanella population genomics, and to understand the evolution, ecophysiology, and speciation of Shewanella. The tasks supporting this aim are: to study genetic and ecophysiological bases defining the core and diversification of Shewanella species; to determine gene content patterns along redox gradients; and to Investigate the evolutionary processes, patterns and mechanisms of Shewanella.

  6. Accurate genome relative abundance estimation based on shotgun metagenomic reads.

    PubMed

    Xia, Li C; Cram, Jacob A; Chen, Ting; Fuhrman, Jed A; Sun, Fengzhu

    2011-01-01

    Accurate estimation of microbial community composition based on metagenomic sequencing data is fundamental for subsequent metagenomics analysis. Prevalent estimation methods are mainly based on directly summarizing alignment results or its variants; often result in biased and/or unstable estimates. We have developed a unified probabilistic framework (named GRAMMy) by explicitly modeling read assignment ambiguities, genome size biases and read distributions along the genomes. Maximum likelihood method is employed to compute Genome Relative Abundance of microbial communities using the Mixture Model theory (GRAMMy). GRAMMy has been demonstrated to give estimates that are accurate and robust across both simulated and real read benchmark datasets. We applied GRAMMy to a collection of 34 metagenomic read sets from four metagenomics projects and identified 99 frequent species (minimally 0.5% abundant in at least 50% of the data-sets) in the human gut samples. Our results show substantial improvements over previous studies, such as adjusting the over-estimated abundance for Bacteroides species for human gut samples, by providing a new reference-based strategy for metagenomic sample comparisons. GRAMMy can be used flexibly with many read assignment tools (mapping, alignment or composition-based) even with low-sensitivity mapping results from huge short-read datasets. It will be increasingly useful as an accurate and robust tool for abundance estimation with the growing size of read sets and the expanding database of reference genomes.

  7. A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells.

    PubMed

    Kunzelmann, Stefan; Böttcher, Romy; Schmidts, Ines; Förstemann, Klaus

    2016-06-01

    Custom genome editing has become an essential element of molecular biology. In particular, the generation of fusion constructs with epitope tags or fluorescent proteins at the genomic locus facilitates the analysis of protein expression, localization, and interaction partners at physiologic levels. Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. While our first PCR-based editing approach offered only blasticidin-resistance for selection, we now introduce puromycin-resistance as a second, independent selection marker; it is thus possible to edit two loci (e.g., for coimmunoprecipitation) without marker removal. Finally, we describe a modified FLP recombinase expression plasmid that improves the efficiency of marker cassette FLP-out. In summary, our technique and reagents enable a flexible, robust, and cloning-free genome editing approach that can be parallelized for scale-up.

  8. InsectBase: a resource for insect genomes and transcriptomes

    PubMed Central

    Yin, Chuanlin; Shen, Gengyu; Guo, Dianhao; Wang, Shuping; Ma, Xingzhou; Xiao, Huamei; Liu, Jinding; Zhang, Zan; Liu, Ying; Zhang, Yiqun; Yu, Kaixiang; Huang, Shuiqing; Li, Fei

    2016-01-01

    The genomes and transcriptomes of hundreds of insects have been sequenced. However, insect community lacks an integrated, up-to-date collection of insect gene data. Here, we introduce the first release of InsectBase, available online at http://www.insect-genome.com. The database encompasses 138 insect genomes, 116 insect transcriptomes, 61 insect gene sets, 36 gene families of 60 insects, 7544 miRNAs of 69 insects, 96 925 piRNAs of Drosophila melanogaster and Chilo suppressalis, 2439 lncRNA of Nilaparvata lugens, 22 536 pathways of 78 insects, 678 881 untranslated regions (UTR) of 84 insects and 160 905 coding sequences (CDS) of 70 insects. This release contains over 12 million sequences and provides search functionality, a BLAST server, GBrowse, insect pathway construction, a Facebook-like network for the insect community (iFacebook), and phylogenetic analysis of selected genes. PMID:26578584

  9. InsectBase: a resource for insect genomes and transcriptomes.

    PubMed

    Yin, Chuanlin; Shen, Gengyu; Guo, Dianhao; Wang, Shuping; Ma, Xingzhou; Xiao, Huamei; Liu, Jinding; Zhang, Zan; Liu, Ying; Zhang, Yiqun; Yu, Kaixiang; Huang, Shuiqing; Li, Fei

    2016-01-04

    The genomes and transcriptomes of hundreds of insects have been sequenced. However, insect community lacks an integrated, up-to-date collection of insect gene data. Here, we introduce the first release of InsectBase, available online at http://www.insect-genome.com. The database encompasses 138 insect genomes, 116 insect transcriptomes, 61 insect gene sets, 36 gene families of 60 insects, 7544 miRNAs of 69 insects, 96,925 piRNAs of Drosophila melanogaster and Chilo suppressalis, 2439 lncRNA of Nilaparvata lugens, 22,536 pathways of 78 insects, 678,881 untranslated regions (UTR) of 84 insects and 160,905 coding sequences (CDS) of 70 insects. This release contains over 12 million sequences and provides search functionality, a BLAST server, GBrowse, insect pathway construction, a Facebook-like network for the insect community (iFacebook), and phylogenetic analysis of selected genes.

  10. Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences

    PubMed Central

    Jelen, Mateja M.; Chen, Zigui; Kocjan, Boštjan J.; Hošnjak, Lea; Burt, Felicity J.; Chan, Paul K. S.; Chouhy, Diego; Combrinck, Catharina E.; Estrade, Christine; Fiander, Alison; Garland, Suzanne M.; Giri, Adriana A.; González, Joaquín Víctor; Gröning, Arndt; Hibbitts, Sam; Luk, Tommy N. M.; Marinic, Karina; Matsukura, Toshihiko; Neumann, Anna; Oštrbenk, Anja; Picconi, Maria Alejandra; Sagadin, Martin; Sahli, Roland; Seedat, Riaz Y.; Seme, Katja; Severini, Alberto; Sinchi, Jessica L.; Smahelova, Jana; Tabrizi, Sepehr N.; Tachezy, Ruth; Tohme Faybush, Sarah; Uloza, Virgilijus; Uloziene, Ingrida; Wong, Yong Wee; Židovec Lepej, Snježana; Burk, Robert D.

    2016-01-01

    ABSTRACT Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B. IMPORTANCE This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay

  11. Use of genome-editing tools to treat sickle cell disease.

    PubMed

    Tasan, Ipek; Jain, Surbhi; Zhao, Huimin

    2016-09-01

    Recent advances in genome-editing techniques have made it possible to modify any desired DNA sequence by employing programmable nucleases. These next-generation genome-modifying tools are the ideal candidates for therapeutic applications, especially for the treatment of genetic disorders like sickle cell disease (SCD). SCD is an inheritable monogenic disorder which is caused by a point mutation in the β-globin gene. Substantial success has been achieved in the development of supportive therapeutic strategies for SCD, but unfortunately there is still a lack of long-term universal cure. The only existing curative treatment is based on allogeneic stem cell transplantation from healthy donors; however, this treatment is applicable to a limited number of patients only. Hence, a universally applicable therapy is highly desirable. In this review, we will discuss the three programmable nucleases that are commonly used for genome-editing purposes: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). We will continue by exemplifying uses of these methods to correct the sickle cell mutation. Additionally, we will present induction of fetal globin expression as an alternative approach to cure sickle cell disease. We will conclude by comparing the three methods and explaining the concerns about their use in therapy.

  12. Modeling Human Bone Marrow Failure Syndromes Using Pluripotent Stem Cells and Genome Engineering.

    PubMed

    Jung, Moonjung; Dunbar, Cynthia E; Winkler, Thomas

    2015-12-01

    The combination of epigenetic reprogramming with advanced genome editing technologies opened a new avenue to study disease mechanisms, particularly of disorders with depleted target tissue. Bone marrow failure syndromes (BMFS) typically present with a marked reduction of peripheral blood cells due to a destroyed or dysfunctional bone marrow compartment. Somatic and germline mutations have been etiologically linked to many cases of BMFS. However, without the ability to study primary patient material, the exact pathogenesis for many entities remained fragmentary. Capturing the pathological genotype in induced pluripotent stem cells (iPSCs) allows studying potential developmental defects leading to a particular phenotype. The lack of hematopoietic stem and progenitor cells in these patients can also be overcome by differentiating patient-derived iPSCs into hematopoietic lineages. With fast growing genome editing techniques, such as CRISPR/Cas9, correction of disease-causing mutations in iPSCs or introduction of mutations in cells from healthy individuals enable comparative studies that may identify other genetic or epigenetic events contributing to a specific disease phenotype. In this review, we present recent progresses in disease modeling of inherited and acquired BMFS using reprogramming and genome editing techniques. We also discuss the challenges and potential shortcomings of iPSC-based models for hematological diseases.

  13. Dissecting diabetes/metabolic disease mechanisms using pluripotent stem cells and genome editing tools

    PubMed Central

    Teo, Adrian Kee Keong; Gupta, Manoj K.; Doria, Alessandro; Kulkarni, Rohit N.

    2015-01-01

    Background Diabetes and metabolic syndromes are chronic, devastating diseases with increasing prevalence. Human pluripotent stem cells are gaining popularity in their usage for human in vitro disease modeling. With recent rapid advances in genome editing tools, these cells can now be genetically manipulated with relative ease to study how genes and gene variants contribute to diabetes and metabolic syndromes. Scope of review We highlight the diabetes and metabolic genes and gene variants, which could potentially be studied, using two powerful technologies – human pluripotent stem cells (hPSCs) and genome editing tools – to aid the elucidation of yet elusive mechanisms underlying these complex diseases. Major conclusions hPSCs and the advancing genome editing tools appear to be a timely and potent combination for probing molecular mechanism(s) underlying diseases such as diabetes and metabolic syndromes. The knowledge gained from these hiPSC-based disease modeling studies can potentially be translated into the clinics by guiding clinicians on the appropriate type of medication to use for each condition based on the mechanism of action of the disease. PMID:26413465

  14. Comprehensive genomic characterization of head and neck squamous cell carcinomas.

    PubMed

    2015-01-29

    The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.

  15. Comprehensive genomic characterization of head and neck squamous cell carcinomas

    PubMed Central

    2014-01-01

    The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. We find that human papillomavirus-associated (HPV) tumors are dominated by helicase domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss of TP53 mutations and CDKN2A with frequent copy number alterations including a novel amplification of 11q22. A subgroup of oral cavity tumors with favorable clinical outcomes displayed infrequent CNAs in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and wild-type TP53. Other distinct subgroups harbored novel loss of function alterations of the chromatin modifier NSD1, Wnt pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumors. Therapeutic candidate alterations were identified in the majority of HNSCC's. PMID:25631445

  16. Site-Specific Genome Engineering in Human Pluripotent Stem Cells

    PubMed Central

    Merkert, Sylvia; Martin, Ulrich

    2016-01-01

    The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies. PMID:27347935

  17. Site-Specific Genome Engineering in Human Pluripotent Stem Cells.

    PubMed

    Merkert, Sylvia; Martin, Ulrich

    2016-06-24

    The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies.

  18. INTEGRATED GENOME-BASED STUDIES OF SHEWANELLA ECOPHYSIOLOGY

    SciTech Connect

    NEALSON, KENNETH H.

    2013-10-15

    laboratories. Applications: 1. Corrosion: Electron flow is often part of the corrosive process, and several studies were done in concert with this proposal with regard to the ability of EET-capable bacteria to enhance, inhibit, or detect corrosion. These included using EET-capable bacteria to detect corrosion in its earliest stages [5], to use corrosion-causing bacteria for the study of the microbe/mineral interface during corrosion [1], and to study the groups of microbes involved with corrosion of natural systems [19]. 2. Bioenergy and microbial fuel cells: The production of electricity by Shewanella was shown early in this program (several years ago) to be dependent on the genes for extracellular electron transport (EET), and applied work involved the testing of various strains and conditions for the optimization of current production by the shewanellae [11,14,16]. 3. Identification of shewanellae strains: Based on similarities seen in genomic comparisons, a rapid method was employed for distinguishing between shewanellae strains [17]. Interactions with other laboratories: This grant was an extension of a grant involving the so-called ?Shewanella Federation?, and as such, a number of our publications were joint with other members of this group. The groups included: 1. Pacific Northwest Laboratories ? 2. Oak Ridge National Labs 3. Michigan State University 4. University of Oklahoma 5. Naval Research Laboratory, Washington DC 6. Burnham Medical Research Institute, San Diego 7. J. Craig Venter Institute, San Diego Education: Graduate Students: Michael Waters, Ph.D. ? at NIST, Washington D.C. Lewis Hsu, Ph.D. ? at NRL, San Diego Howard Harris, Ph.D. ? Postdoc at University, France Everett Salas, Ph.D. ? Scientist at Chevron McLean, Jeffrey, Ph.D. ? Scientist at J. Craig Venter Institute McCrow, John, Ph.D. ? Scientist at J. Craig Venter Institute Postdocs: Mohamed El-Naggar ? Professor of Physics, USC Jinjun Kan ? Senior Researcher at Undergraduatges: During this year, we had

  19. Maintenance of Genome Integrity: How Mammalian Cells Orchestrate Genome Duplication by Coordinating Replicative and Specialized DNA Polymerases

    PubMed Central

    Barnes, Ryan; Eckert, Kristin

    2017-01-01

    Precise duplication of the human genome is challenging due to both its size and sequence complexity. DNA polymerase errors made during replication, repair or recombination are central to creating mutations that drive cancer and aging. Here, we address the regulation of human DNA polymerases, specifically how human cells orchestrate DNA polymerases in the face of stress to complete replication and maintain genome stability. DNA polymerases of the B-family are uniquely adept at accurate genome replication, but there are numerous situations in which one or more additional DNA polymerases are required to complete genome replication. Polymerases of the Y-family have been extensively studied in the bypass of DNA lesions; however, recent research has revealed that these polymerases play important roles in normal human physiology. Replication stress is widely cited as contributing to genome instability, and is caused by conditions leading to slowed or stalled DNA replication. Common Fragile Sites epitomize “difficult to replicate” genome regions that are particularly vulnerable to replication stress, and are associated with DNA breakage and structural variation. In this review, we summarize the roles of both the replicative and Y-family polymerases in human cells, and focus on how these activities are regulated during normal and perturbed genome replication. PMID:28067843

  20. Maintenance of Genome Integrity: How Mammalian Cells Orchestrate Genome Duplication by Coordinating Replicative and Specialized DNA Polymerases.

    PubMed

    Barnes, Ryan; Eckert, Kristin

    2017-01-06

    Precise duplication of the human genome is challenging due to both its size and sequence complexity. DNA polymerase errors made during replication, repair or recombination are central to creating mutations that drive cancer and aging. Here, we address the regulation of human DNA polymerases, specifically how human cells orchestrate DNA polymerases in the face of stress to complete replication and maintain genome stability. DNA polymerases of the B-family are uniquely adept at accurate genome replication, but there are numerous situations in which one or more additional DNA polymerases are required to complete genome replication. Polymerases of the Y-family have been extensively studied in the bypass of DNA lesions; however, recent research has revealed that these polymerases play important roles in normal human physiology. Replication stress is widely cited as contributing to genome instability, and is caused by conditions leading to slowed or stalled DNA replication. Common Fragile Sites epitomize "difficult to replicate" genome regions that are particularly vulnerable to replication stress, and are associated with DNA breakage and structural variation. In this review, we summarize the roles of both the replicative and Y-family polymerases in human cells, and focus on how these activities are regulated during normal and perturbed genome replication.

  1. Combining magnetic sorting of mother cells and fluctuation tests to analyze genome instability during mitotic cell aging in Saccharomyces cerevisiae.

    PubMed

    Patterson, Melissa N; Maxwell, Patrick H

    2014-10-16

    Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on

  2. CRISPR-based screening of genomic island excision events in bacteria.

    PubMed

    Selle, Kurt; Klaenhammer, Todd R; Barrangou, Rodolphe

    2015-06-30

    Genomic analysis of Streptococcus thermophilus revealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats-CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPR-Cas systems and MGEs is one of the driving forces governing genome homeostasis in this genus. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs, in silico prediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. Targeting lacZ within the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac(-) survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.

  3. Effect of amino acid supplementation on titer and glycosylation distribution in hybridoma cell cultures-Systems biology-based interpretation using genome-scale metabolic flux balance model and multivariate data analysis.

    PubMed

    Reimonn, Thomas M; Park, Seo-Young; Agarabi, Cyrus D; Brorson, Kurt A; Yoon, Seongkyu

    2016-09-01

    Genome-scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745-753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome-scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163-1173, 2016.

  4. Mutagenesis and Genome Engineering of Epstein-Barr Virus in Cultured Human Cells by CRISPR/Cas9.

    PubMed

    Yuen, Kit-San; Chan, Chi-Ping; Kok, Kin-Hang; Jin, Dong-Yan

    2017-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein-Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.

  5. De novo DNA methylation of the paternal genome in 2-cell mouse embryos.

    PubMed

    Ma, X S; Wang, X G; Qin, L; Song, C L; Lin, F; Song, J M; Zhu, C C; Liu, H L

    2014-10-27

    The developmental dynamics of DNA methylation events have been well studied. Active demethylation of the paternal genome occurs in the zygote, passive demethylation occurs during cleavage stages, and de novo methylation occurs by the blastocyst stage. It is believed that the paternal genome has lower levels of methylation during early development than the maternal genome. However, in this study, we provide direct and indirect evidence of genome-wide de novo DNA methylation of the paternal genome after the first cell cycle in mouse embryos. Although very little methylation was detected within the male pronucleus in zygotes, an intense methylation signal was clearly visible within the androgenetic 2-cell embryos. Moreover, the DNA methylation level of the paternal genome in the post-zygotic metaphase embryos was similar to that of the maternal genome. Using indirect immunofluorescence with an antibody to methylated lysine 9 in histone H3, we provided new evidence to support the concept of spatial compartmentalization of parental genomes in 2-cell mouse embryos. Nevertheless, the transient segregation of parental genomes was not observed by determining the DNA methylation distribution in the 2-cell embryos even though DNA methylation asymmetry between the maternal and paternal pronucleus existed in the 1-cell stage. The disappearance of separate immunofluorescence signals of 5-methyl cytosine in the 2-cell embryos might be attributed to the de novo methylation of the paternal genome during the first mitotic cycle.

  6. Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability.

    PubMed

    Patterson, Melissa N; Scannapieco, Alison E; Au, Pak Ho; Dorsey, Savanna; Royer, Catherine A; Maxwell, Patrick H

    2015-10-01

    Retrotransposon expression or mobility is increased with age in multiple species and could promote genome instability or altered gene expression during aging. However, it is unclear whether activation of retrotransposons during aging is an indirect result of global changes in chromatin and gene regulation or a result of retrotransposon-specific mechanisms. Retromobility of a marked chromosomal Ty1 retrotransposon in Saccharomyces cerevisiae was elevated in mother cells relative to their daughter cells, as determined by magnetic cell sorting of mothers and daughters. Retromobility frequencies in aging mother cells were significantly higher than those predicted by cell age and the rate of mobility in young populations, beginning when mother cells were only several generations old. New Ty1 insertions in aging mothers were more strongly correlated with gross chromosome rearrangements than in young cells and were more often at non-preferred target sites. Mother cells were more likely to have high concentrations and bright foci of Ty1 Gag-GFP than their daughter cells. Levels of extrachromosomal Ty1 cDNA were also significantly higher in aged mother cell populations than their daughter cell populations. These observations are consistent with a retrotransposon-specific mechanism that causes retrotransposition to occur preferentially in yeast mother cells as they begin to age, as opposed to activation by phenotypic changes associated with very old age. These findings will likely be relevant for understanding retrotransposons and aging in many organisms, based on similarities in regulation and consequences of retrotransposition in diverse species.

  7. Population genetics inside a cell: Mutations and mitochondrial genome maintenance

    NASA Astrophysics Data System (ADS)

    Goyal, Sidhartha; Shraiman, Boris; Gottschling, Dan

    2012-02-01

    In realistic ecological and evolutionary systems natural selection acts on multiple levels, i.e. it acts on individuals as well as on collection of individuals. An understanding of evolutionary dynamics of such systems is limited in large part due to the lack of experimental systems that can challenge theoretical models. Mitochondrial genomes (mtDNA) are subjected to selection acting on cellular as well as organelle levels. It is well accepted that mtDNA in yeast Saccharomyces cerevisiae is unstable and can degrade over time scales comparable to yeast cell division time. We utilize a recent technology designed in Gottschling lab to extract DNA from populations of aged yeast cells and deep sequencing to characterize mtDNA variation in a population of young and old cells. In tandem, we developed a stochastic model that includes the essential features of mitochondrial biology that provides a null model for expected mtDNA variation. Overall, we find approximately 2% of the polymorphic loci that show significant increase in frequency as cells age providing direct evidence for organelle level selection. Such quantitative study of mtDNA dynamics is absolutely essential to understand the propagation of mtDNA mutations linked to a spectrum of age-related diseases in humans.

  8. Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues

    PubMed Central

    Frumkin, Dan; Wasserstrom, Adam; Itzkovitz, Shalev; Harmelin, Alon; Rechavi, Gideon; Shapiro, Ehud

    2008-01-01

    Background Whole genome amplification (WGA) and laser assisted micro-dissection represent two recently developed technologies that can greatly advance biological and medical research. WGA allows the analysis of multiple genomic loci from a single genome and has been performed on single cells from cell suspensions and from enzymatically-digested tissues. Laser micro-dissection makes it possible to isolate specific single cells from heterogeneous tissues. Results Here we applied for the first time WGA on laser micro-dissected single cells from stained tissue sections, and developed a protocol for sequentially performing the two procedures. The combined procedure allows correlating the cell's genome with its natural morphology and precise anatomical position. From each cell we amplified 122 genomic and mitochondrial loci. In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to ~700000 copies each, and mitochondrial DNA was amplified successfully in all cells. Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results. Sequencing and capillary electrophoresis of WGA products allowed detection of slippage mutations in microsatellites (MS), and point mutations in P53. Conclusion Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays. PMID:18284708

  9. Cre Fused with RVG Peptide Mediates Targeted Genome Editing in Mouse Brain Cells In Vivo

    PubMed Central

    Zou, Zhiyuan; Sun, Zhaolin; Li, Pan; Feng, Tao; Wu, Sen

    2016-01-01

    Cell penetrating peptides (CPPs) are short peptides that can pass through cell membranes. CPPs can facilitate the cellular entry of proteins, macromolecules, nanoparticles and drugs. RVG peptide (RVG hereinafter) is a 29-amino-acid CPP derived from a rabies virus glycoprotein that can cross the blood-brain barrier (BBB) and enter brain cells. However, whether RVG can be used for genome editing in the brain has not been reported. In this work, we combined RVG with Cre recombinase for bacterial expression. The purified RVG-Cre protein cut plasmids in vitro and traversed cell membranes in cultured Neuro2a cells. By tail vein-injecting RVG-Cre into Cre reporter mouse lines mTmG and Rosa26lacZ, we demonstrated that RVG-Cre could target brain cells and achieve targeted somatic genome editing in adult mice. This direct delivery of the gene-editing enzyme protein into mouse brains with RVG is much safer than plasmid- or viral-based methods, holding promise for further applications in the treatment of various brain diseases. PMID:27983648

  10. Development of Advanced Technologies for Complete Genomic and Proteomic Characterization of Quantized Human Tumor Cells

    DTIC Science & Technology

    2015-09-01

    populations were successfully established from the corresponding parental cell lines (Figure 2). To generate quantized cell populations a single ...individual cells from the SN291 parental culture. Each dot represents a single cell. Color gradient indicates enrichment score for either published CD133... parental lines and quantized cell types (Specific Aim 5). We believe this program has significantly advanced genomic, proteomic and single -cell

  11. Prostate Cancer Diagnostics and Prognostics Based on Interphase Spatial Genome Positioning

    DTIC Science & Technology

    2016-03-01

    architecture in quiescent and senescent human fibroblasts. Curr Biol 10, 149–152. Cooperberg MR, Broering JM, Carroll PR (2010). Time trends and local...10.1016/j.cell.2012.09.043. 832 Spatial Genome Organization based Diagnostics 20 Cooperberg, M.R., Broering, J.M., and Carroll , P.R. (2009). Risk...835 Cooperberg, M.R., Broering, J.M., and Carroll , P.R. (2010). Time trends and local variation in 836 primary treatment of localized prostate

  12. Fluorescence-based resource for semi-automated genomic analyses

    SciTech Connect

    Kiser, M.B.; Dragwa, C.; Jedlicka, A.E.

    1994-09-01

    To facilitate the practical application of highly efficient semi-automated methods for general application in genomic analyses, we have developed a fluorescence-based marker resource. Ninety highly polymorphic simple tandem repeat markers were combined to provide a rapid, accurate, and highly efficient initial genome-wide screening system. These markers are spaced on average every 33 recombination units, with a mean heterozygosity of 81% (range 65-94%), covering 22 autosomes and the X and Y chromosomes. Less than 3% of the genome lies beyond 30 cM of the nearest marker. Markers were placed in a vertical ladder that we have termed a SET according to the size of the PCR fragments they produce during electrophoresis. Each SET was designed to avoid overlap between loci during gel separations to assure accuracy when scoring genotypes. We have constructed 15 SETS of markers. Three SETS, each labelled with one of three fluors, were combined into what we have termed a GROUP, which is co-electrophoresed with internal size standards that are labelled with a fourth flour. Five GROUPS of markers were assembled that contain a total of 15 SETS of markers. Each GROUP cover 18 regions of the genome that can be detected simultaneously, since this genomic analysis system is fully compatible with automated fragment analyzers using simultaneous four-color fluorescence-based detection systems. This allows for multiplex detection and a throughput of 1,944 genotypes daily per instrument. This system will be highly beneficial in a number of clinical and research applications including: linkage, cancer genetics, forensics, and cytogenetics.

  13. A web-based genomic sequence database for the Streptomycetaceae: a tool for systematics and genome mining

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ARS Microbial Genome Sequence Database (http://199.133.98.43), a web-based database server, was established utilizing the BIGSdb (Bacterial Isolate Genomics Sequence Database) software package, developed at Oxford University, as a tool to manage multi-locus sequence data for the family Streptomy...

  14. Natural Biased Coin Encoded in the Genome Determines Cell Strategy

    PubMed Central

    Dorri, Faezeh; Mahini, Hamid; Sharifi-Zarchi, Ali; Totonchi, Mehdi; Tusserkani, Ruzbeh; Pezeshk, Hamid; Sadeghi, Mehdi

    2014-01-01

    Decision making at a cellular level determines different fates for isogenic cells. However, it is not yet clear how rational decisions are encoded in the genome, how they are transmitted to their offspring, and whether they evolve and become optimized throughout generations. In this paper, we use a game theoretic approach to explain how rational decisions are made in the presence of cooperators and competitors. Our results suggest the existence of an internal switch that operates as a biased coin. The biased coin is, in fact, a biochemical bistable network of interacting genes that can flip to one of its stable states in response to different environmental stimuli. We present a framework to describe how the positions of attractors in such a gene regulatory network correspond to the behavior of a rational player in a competing environment. We evaluate our model by considering lysis/lysogeny decision making of bacteriophage lambda in E. coli. PMID:25090629

  15. Targeted gene correction minimally impacts whole-genome mutational load in human-disease-specific induced pluripotent stem cell clones.

    PubMed

    Suzuki, Keiichiro; Yu, Chang; Qu, Jing; Li, Mo; Yao, Xiaotian; Yuan, Tingting; Goebl, April; Tang, Senwei; Ren, Ruotong; Aizawa, Emi; Zhang, Fan; Xu, Xiuling; Soligalla, Rupa Devi; Chen, Feng; Kim, Jessica; Kim, Na Young; Liao, Hsin-Kai; Benner, Chris; Esteban, Concepcion Rodriguez; Jin, Yabin; Liu, Guang-Hui; Li, Yingrui; Izpisua Belmonte, Juan Carlos

    2014-07-03

    The utility of genome editing technologies for disease modeling and developing cellular therapies has been extensively documented, but the impact of these technologies on mutational load at the whole-genome level remains unclear. We performed whole-genome sequencing to evaluate the mutational load at single-base resolution in individual gene-corrected human induced pluripotent stem cell (hiPSC) clones in three different disease models. In single-cell clones, gene correction by helper-dependent adenoviral vector (HDAdV) or Transcription Activator-Like Effector Nuclease (TALEN) exhibited few off-target effects and a low level of sequence variation, comparable to that accumulated in routine hiPSC culture. The sequence variants were randomly distributed and unique to individual clones. We also combined both technologies and developed a TALEN-HDAdV hybrid vector, which significantly increased gene-correction efficiency in hiPSCs. Therefore, with careful monitoring via whole-genome sequencing it is possible to apply genome editing to human pluripotent cells with minimal impact on genomic mutational load.

  16. The Karyote physico-chemical genomic, proteomic, metabolic cell modeling system.

    PubMed

    Ortoleva, P; Berry, E; Brun, Y; Fan, J; Fontus, M; Hubbard, K; Jaqaman, K; Jarymowycz, L; Navid, A; Sayyed-Ahmad, A; Shreif, Z; Stanley, F; Tuncay, K; Weitzke, E; Wu, L-C

    2003-01-01

    Modeling approaches to the dynamics of a living cell are presented that are strongly based on its underlying physical and chemical processes and its hierarchical spatio-temporal organization. Through the inclusion of a broad spectrum of processes and a rigorous analysis of the multiple scale nature of cellular dynamics, we are attempting to advance cell modeling and its applications. The presentation focuses on our cell modeling system, which integrates data archiving and quantitative physico-chemical modeling and information theory to provide a seamless approach to the modeling/data analysis endeavor. Thereby the rapidly growing mess of genomic, proteomic, metabolic, and cell physiological data can be automatically used to develop and calibrate a predictive cell model. The discussion focuses on the Karyote cell modeling system and an introduction to the CellX and VirusX models. The Karyote software system integrates three elements: (1) a model-building and data archiving module that allows one to define a cell type to be modeled through its reaction network, structure, and transport processes as well as to choose the surrounding medium and other parameters of the phenomenon to be modeled; (2) a genomic, proteomic, metabolic cell simulator that solves the equations of metabolic reaction, transcription/translation polymerization and the exchange of molecules between parts of the cell and with the surrounding medium; and (3) an information theory module (ITM) that automates model calibration and development, and integrates a variety of data types with the cell dynamic computations. In Karyote, reactions may be fast (equilibrated) or slow (finite rate), and the special effects of enzymes and other minority species yielding steady-state cycles of arbitrary complexities are accounted for. These features of the dynamics are handled via rigorous multiple scale analysis. A user interface allows for an automated generation and solution of the equations of multiple timescale

  17. Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

    SciTech Connect

    Angelova, Angelina; Park, Sang-Hycuk; Kyndt, John; Fitzsimmons, Kevin; Brown, Judith K

    2013-09-01

    With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.

  18. Micro and nanofluidic structures for cell sorting and genomic analysis

    NASA Astrophysics Data System (ADS)

    Morton, Keith J.

    Microfluidic systems promise rapid analysis of small samples in a compact and inexpensive format. But direct scaling of lab bench protocols on-chip is challenging because laminar flows in typical microfluidic devices are characterized by non-mixing streamlines. Common microfluidic mixers and sorters work by diffusion, limiting application to objects that diffuse slowly such as cells and DNA. Recently Huang et.al. developed a passive microfluidic element to continuously separate bio-particles deterministically. In Deterministic Lateral Displacement (DLD), objects are sorted by size as they transit an asymmetric array of microfabricated posts. This thesis further develops DLD arrays with applications in three broad new areas. First the arrays are used, not simply to sort particles, but to move streams of cells through functional flows for chemical treatment---such as on-chip immunofluorescent labeling of blood cells with washing, and on-chip E.coli cell lysis with simultaneous chromosome extraction. Secondly, modular tiling of the basic DLD element is used to construct complex particle handling modes that include beam steering for jets of cells and beads. Thirdly, nanostructured DLD arrays are built using Nanoimprint Lithography (NIL) and continuous-flow separation of 100 nm and 200 nm size particles is demonstrated. Finally a number of ancillary nanofabrication techniques were developed in support of these overall goals, including methods to interface nanofluidic structures with standard microfluidic components such as inlet channels and reservoirs, precision etching of ultra-high aspect ratio (>50:1) silicon nanostructures, and fabrication of narrow (˜ 35 nm) channels used to stretch genomic length DNA.

  19. The Comprehensive Phytopathogen Genomics Resource: a web-based resource for data-mining plant pathogen genomes.

    PubMed

    Hamilton, John P; Neeno-Eckwall, Eric C; Adhikari, Bishwo N; Perna, Nicole T; Tisserat, Ned; Leach, Jan E; Lévesque, C André; Buell, C Robin

    2011-01-01

    The Comprehensive Phytopathogen Genomics Resource (CPGR) provides a web-based portal for plant pathologists and diagnosticians to view the genome and trancriptome sequence status of 806 bacterial, fungal, oomycete, nematode, viral and viroid plant pathogens. Tools are available to search and analyze annotated genome sequences of 74 bacterial, fungal and oomycete pathogens. Oomycete and fungal genomes are obtained directly from GenBank, whereas bacterial genome sequences are downloaded from the A Systematic Annotation Package (ASAP) database that provides curation of genomes using comparative approaches. Curated lists of bacterial genes relevant to pathogenicity and avirulence are also provided. The Plant Pathogen Transcript Assemblies Database provides annotated assemblies of the transcribed regions of 82 eukaryotic genomes from publicly available single pass Expressed Sequence Tags. Data-mining tools are provided along with tools to create candidate diagnostic markers, an emerging use for genomic sequence data in plant pathology. The Plant Pathogen Ribosomal DNA (rDNA) database is a resource for pathogens that lack genome or transcriptome data sets and contains 131 755 rDNA sequences from GenBank for 17 613 species identified as plant pathogens and related genera. Database URL: http://cpgr.plantbiology.msu.edu.

  20. Single-cell genomics reveal low recombination frequencies in freshwater bacteria of the SAR11 clade

    PubMed Central

    2013-01-01

    Background The SAR11 group of Alphaproteobacteria is highly abundant in the oceans. It contains a recently diverged freshwater clade, which offers the opportunity to compare adaptations to salt- and freshwaters in a monophyletic bacterial group. However, there are no cultivated members of the freshwater SAR11 group and no genomes have been sequenced yet. Results We isolated ten single SAR11 cells from three freshwater lakes and sequenced and assembled their genomes. A phylogeny based on 57 proteins indicates that the cells are organized into distinct microclusters. We show that the freshwater genomes have evolved primarily by the accumulation of nucleotide substitutions and that they have among the lowest ratio of recombination to mutation estimated for bacteria. In contrast, members of the marine SAR11 clade have one of the highest ratios. Additional metagenome reads from six lakes confirm low recombination frequencies for the genome overall and reveal lake-specific variations in microcluster abundances. We identify hypervariable regions with gene contents broadly similar to those in the hypervariable regions of the marine isolates, containing genes putatively coding for cell surface molecules. Conclusions We conclude that recombination rates differ dramatically in phylogenetic sister groups of the SAR11 clade adapted to freshwater and marine ecosystems. The results suggest that the transition from marine to freshwater systems has purged diversity and resulted in reduced opportunities for recombination with divergent members of the clade. The low recombination frequencies of the LD12 clade resemble the low genetic divergence of host-restricted pathogens that have recently shifted to a new host. PMID:24286338

  1. The genomic and transcriptomic landscape of a HeLa cell line.

    PubMed

    Landry, Jonathan J M; Pyl, Paul Theodor; Rausch, Tobias; Zichner, Thomas; Tekkedil, Manu M; Stütz, Adrian M; Jauch, Anna; Aiyar, Raeka S; Pau, Gregoire; Delhomme, Nicolas; Gagneur, Julien; Korbel, Jan O; Huber, Wolfgang; Steinmetz, Lars M

    2013-08-07

    HeLa is the most widely used model cell line for studying human cellular and molecular biology. To date, no genomic reference for this cell line has been released, and experiments have relied on the human reference genome. Effective design and interpretation of molecular genetic studies performed using HeLa cells require accurate genomic information. Here we present a detailed genomic and transcriptomic characterization of a HeLa cell line. We performed DNA and RNA sequencing of a HeLa Kyoto cell line and analyzed its mutational portfolio and gene expression profile. Segmentation of the genome according to copy number revealed a remarkably high level of aneuploidy and numerous large structural variants at unprecedented resolution. Some of the extensive genomic rearrangements are indicative of catastrophic chromosome shattering, known as chromothripsis. Our analysis of the HeLa gene expression profile revealed that several pathways, including cell cycle and DNA repair, exhibit significantly different expression patterns from those in normal human tissues. Our results provide the first detailed account of genomic variants in the HeLa genome, yielding insight into their impact on gene expression and cellular function as well as their origins. This study underscores the importance of accounting for the strikingly aberrant characteristics of HeLa cells when designing and interpreting experiments, and has implications for the use of HeLa as a model of human biology.

  2. Marker-Based Estimation of Genetic Parameters in Genomics

    PubMed Central

    Hu, Zhiqiu; Yang, Rong-Cai

    2014-01-01

    Linear mixed model (LMM) analysis has been recently used extensively for estimating additive genetic variances and narrow-sense heritability in many genomic studies. While the LMM analysis is computationally less intensive than the Bayesian algorithms, it remains infeasible for large-scale genomic data sets. In this paper, we advocate the use of a statistical procedure known as symmetric differences squared (SDS) as it may serve as a viable alternative when the LMM methods have difficulty or fail to work with large datasets. The SDS procedure is a general and computationally simple method based only on the least squares regression analysis. We carry out computer simulations and empirical analyses to compare the SDS procedure with two commonly used LMM-based procedures. Our results show that the SDS method is not as good as the LMM methods for small data sets, but it becomes progressively better and can match well with the precision of estimation by the LMM methods for data sets with large sample sizes. Its major advantage is that with larger and larger samples, it continues to work with the increasing precision of estimation while the commonly used LMM methods are no longer able to work under our current typical computing capacity. Thus, these results suggest that the SDS method can serve as a viable alternative particularly when analyzing ‘big’ genomic data sets. PMID:25025305

  3. Genome-based bioprospecting of microbes for new therapeutics.

    PubMed

    Zotchev, Sergey B; Sekurova, Olga N; Katz, Leonard

    2012-12-01

    Bioprospecting of natural sources for new medicines has a long and successful history, exemplified by the fact that over 50% of all drugs currently on the market are either derived from or inspired by natural products. However, development of new natural product-based therapeutics has been on the decline over the past 20 years, mainly owing to frequent re-discovery of already known compounds coupled with high costs for screening, characterization and development. With the onset of the genomic era allowing rapid sequencing and analysis of bacterial and fungal genomes, it became evident that these organisms possess 'hidden treasures' in the form of gene clusters potentially governing biosynthesis of novel biologically active compounds. This review highlights current progress in mining for and expression of these gene clusters, which may revolutionize the drug discovery pipelines in the near future.

  4. Genomic and epigenetic insights into the molecular bases of heterosis.

    PubMed

    Chen, Z Jeffrey

    2013-07-01

    Heterosis, also known as hybrid vigour, is widespread in plants and animals, but the molecular bases for this phenomenon remain elusive. Recent studies in hybrids and allopolyploids using transcriptomic, proteomic, metabolomic, epigenomic and systems biology approaches have provided new insights. Emerging genomic and epigenetic perspectives suggest that heterosis arises from allelic interactions between parental genomes, leading to altered programming of genes that promote the growth, stress tolerance and fitness of hybrids. For example, epigenetic modifications of key regulatory genes in hybrids and allopolyploids can alter complex regulatory networks of physiology and metabolism, thus modulating biomass and leading to heterosis. The conceptual advances could help to improve plant and animal productivity through the manipulation of heterosis.

  5. The genomic landscape of histone modifications in human T cells

    PubMed Central

    Roh, Tae-Young; Cuddapah, Suresh; Cui, Kairong; Zhao, Keji

    2006-01-01

    To understand the molecular basis that supports the dynamic gene expression programs unique to T cells, we investigated the genomic landscape of activating histone modifications, including histone H3 K9/K14 diacetylation (H3K9acK14ac), H3 K4 trimethylation (H3K4me3), and the repressive histone modification H3 K27 trimethylation (H3K27me3) in primary human T cells. We show that H3K9acK14ac and H3K4me3 are associated with active genes required for T cell function and development, whereas H3K27me3 is associated with silent genes that are involved in development in other cell types. Unexpectedly, we find that 3,330 gene promoters are associated with all of these histone modifications. The gene expression levels are correlated with both the absolute and relative levels of the activating H3K4me3 and the repressive H3K27me3 modifications. Our data reveal that rapidly inducible genes are associated with the H3 acetylation and H3K4me3 modifications, suggesting they assume a chromatin structure poised for activation. In addition, we identified a subpopulation of chromatin regions that are associated with high levels of H3K4me3 and H3K27me3 but low levels of H3K9acK14ac. Therefore, these regions have a distinctive chromatin modification pattern and thus may represent a distinct class of chromatin domains. PMID:17043231

  6. Genomic Alteration in Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Inferred from Karyotyping, Molecular Cytogenetics, and Array Comparative Genomic Hybridization

    PubMed Central

    Rerkarmnuaychoke, Budsaba; Suntronpong, Aorarat; Fu, Beiyuan; Bodhisuwan, Winai; Peyachoknagul, Surin; Yang, Fengtang; Koontongkaew, Sittichai; Srikulnath, Kornsorn

    2016-01-01

    Genomic alteration in head and neck squamous cell carcinoma (HNSCC) was studied in two cell line pairs (HN30-HN31 and HN4-HN12) using conventional C-banding, multiplex fluorescence in situ hybridization (M-FISH), and array comparative genomic hybridization (array CGH). HN30 and HN4 were derived from primary lesions in the pharynx and base of tongue, respectively, and HN31 and HN12 were derived from lymph-node metastatic lesions belonging to the same patients. Gain of chromosome 1, 7, and 11 were shared in almost all cell lines. Hierarchical clustering revealed that HN31 was closely related to HN4, which shared eight chromosome alteration cases. Large C-positive heterochromatins were found in the centromeric region of chromosome 9 in HN31 and HN4, which suggests complex structural amplification of the repetitive sequence. Array CGH revealed amplification of 7p22.3p11.2, 8q11.23q12.1, and 14q32.33 in all cell lines involved with tumorigenesis and inflammation genes. The amplification of 2p21 (SIX3), 11p15.5 (H19), and 11q21q22.3 (MAML2, PGR, TRPC6, and MMP family) regions, and deletion of 9p23 (PTPRD) and 16q23.1 (WWOX) regions were identified in HN31 and HN12. Interestingly, partial loss of PTPRD (9p23) and WWOX (16q23.1) genes was identified in HN31 and HN12, and the level of gene expression tended to be the down-regulation of PTPRD, with no detectable expression of the WWOX gene. This suggests that the scarcity of PTPRD and WWOX genes might have played an important role in progression of HNSCC, and could be considered as a target for cancer therapy or a biomarker in molecular pathology. PMID:27501229

  7. Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing | Office of Cancer Genomics

    Cancer.gov

    Abstract: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer comprising at least two molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease.

  8. Squamous cell lung cancer: from tumor genomics to cancer therapeutics.

    PubMed

    Gandara, David R; Hammerman, Peter S; Sos, Martin L; Lara, Primo N; Hirsch, Fred R

    2015-05-15

    Squamous cell lung cancer (SCC) represents an area of unmet need in lung cancer research. For the past several years, therapeutic progress in SCC has lagged behind the now more common non-small cell lung cancer histologic subtype of adenocarcinoma. However, recent efforts to define the complex biology underlying SCC have begun to bear fruit in a multitude of ways, including characterization of previously unknown genomic and signaling pathways, delineation of new, potentially actionable molecular targets, and subsequent development of a large number of agents directed against unique SCC-associated molecular abnormalities. For the first time, SCC-specific prognostic gene signatures and predictive biomarkers of new therapeutic agents are emerging. In addition, recent and ongoing clinical trials, including the Lung-MAP master protocol, have been designed to facilitate approval of targeted therapy-biomarker combinations. In this comprehensive review, we describe the current status of SCC therapeutics, recent advances in the understanding of SCC biology and prognostic gene signatures, and the development of innovative new clinical trials, all of which offer new hope for patients with advanced SCC.

  9. p53-Dependent suppression of genome instability in germ cells.

    PubMed

    Otozai, Shinji; Ishikawa-Fujiwara, Tomoko; Oda, Shoji; Kamei, Yasuhiro; Ryo, Haruko; Sato, Ayuko; Nomura, Taisei; Mitani, Hiroshi; Tsujimura, Tohru; Inohara, Hidenori; Todo, Takeshi

    2014-02-01

    Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2(-/-) males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2(-/-) and wild-type fish. By contrast, irradiated p53(-/-) fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2(-/-) fish, but negligible levels in p53(-/-) fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

  10. A method for detecting significant genomic regions associated with oral squamous cell carcinoma using aCGH.

    PubMed

    Kim, Ki-Yeol; Kim, Jin; Kim, Hyung Jun; Nam, Woong; Cha, In-Ho

    2010-05-01

    Array comparative genomic hybridization (aCGH) provides a genome-wide technique for identifying chromosomal aberrations in human diseases, including cancer. Chromosomal aberrations in cancers are defined as regions that contain an increased or decreased DNA copy number, relative to normal samples. The identification of genomic regions associated with systematic aberrations provides insights into initiation and progression of cancer, and improves diagnosis, prognosis, and therapy strategies. The McNemar test can be used to detect differentially expressed genes after discretization of gene expressions in a microarray experiment for the matched dataset. In this study, we propose a method to detect significantly altered DNA regions, shifted McNemar test, which is based on the standard McNemar test and takes into account changes in copy number variations and the region size throughout the whole genome. In addition, this novel method can be used to detect genomic regions associated with the progress of oral squamous cell carcinoma (OSCC). The performance of the proposed method was evaluated based on the homogeneity within the selected regions and the classification accuracies of the selected regions. This method might be useful for identifying new candidate genes that neighbor known genes based on the whole-genomic variation because it detects significant chromosomal regions, not independent probes.

  11. Genome-wide analysis of LXRα activation reveals new transcriptional networks in human atherosclerotic foam cells.

    PubMed

    Feldmann, Radmila; Fischer, Cornelius; Kodelja, Vitam; Behrens, Sarah; Haas, Stefan; Vingron, Martin; Timmermann, Bernd; Geikowski, Anne; Sauer, Sascha

    2013-04-01

    Increased physiological levels of oxysterols are major risk factors for developing atherosclerosis and cardiovascular disease. Lipid-loaded macrophages, termed foam cells, are important during the early development of atherosclerotic plaques. To pursue the hypothesis that ligand-based modulation of the nuclear receptor LXRα is crucial for cell homeostasis during atherosclerotic processes, we analysed genome-wide the action of LXRα in foam cells and macrophages. By integrating chromatin immunoprecipitation-sequencing (ChIP-seq) and gene expression profile analyses, we generated a highly stringent set of 186 LXRα target genes. Treatment with the nanomolar-binding ligand T0901317 and subsequent auto-regulatory LXRα activation resulted in sequence-dependent sharpening of the genome-binding patterns of LXRα. LXRα-binding loci that correlated with differential gene expression revealed 32 novel target genes with potential beneficial effects, which in part explained the implications of disease-associated genetic variation data. These observations identified highly integrated LXRα ligand-dependent transcriptional networks, including the APOE/C1/C4/C2-gene cluster, which contribute to the reversal of cholesterol efflux and the dampening of inflammation processes in foam cells to prevent atherogenesis.

  12. TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity.

    PubMed

    Mussolino, Claudio; Alzubi, Jamal; Fine, Eli J; Morbitzer, Robert; Cradick, Thomas J; Lahaye, Thomas; Bao, Gang; Cathomen, Toni

    2014-06-01

    Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNA-guided endonucleases has been assessed to some extent, little data are available for transcription activator-like effector-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15-30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation.

  13. Prioritization of anticancer drugs against a cancer using genomic features of cancer cells: A step towards personalized medicine

    PubMed Central

    Gupta, Sudheer; Chaudhary, Kumardeep; Kumar, Rahul; Gautam, Ankur; Nanda, Jagpreet Singh; Dhanda, Sandeep Kumar; Brahmachari, Samir Kumar; Raghava, Gajendra P. S.

    2016-01-01

    In this study, we investigated drug profile of 24 anticancer drugs tested against a large number of cell lines in order to understand the relation between drug resistance and altered genomic features of a cancer cell line. We detected frequent mutations, high expression and high copy number variations of certain genes in both drug resistant cell lines and sensitive cell lines. It was observed that a few drugs, like Panobinostat, are effective against almost all types of cell lines, whereas certain drugs are effective against only a limited type of cell lines. Tissue-specific preference of drugs was also seen where a drug is more effective against cell lines belonging to a specific tissue. Genomic features based models have been developed for each anticancer drug and achieved average correlation between predicted and actual growth inhibition of cell lines in the range of 0.43 to 0.78. We hope, our study will throw light in the field of personalized medicine, particularly in designing patient-specific anticancer drugs. In order to serve the scientific community, a webserver, CancerDP, has been developed for predicting priority/potency of an anticancer drug against a cancer cell line using its genomic features (http://crdd.osdd.net/raghava/cancerdp/). PMID:27030518

  14. Heteropolymeric triplex-based genomic assay to detect pathogens or single-nucleotide polymorphisms in human genomic samples.

    PubMed

    Daksis, Jasmine I; Erikson, Glen H

    2007-03-21

    Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP), without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are "canonical triplexes". Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays.

  15. Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

    DOE PAGES

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-11-21

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We reportmore » distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). In conclusion, our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.« less

  16. Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

    SciTech Connect

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-11-21

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). In conclusion, our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.

  17. Cell context dependent p53 genome-wide binding patterns and enrichment at repeats.

    PubMed

    Botcheva, Krassimira; McCorkle, Sean R

    2014-01-01

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). Our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.

  18. Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

    PubMed Central

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-01-01

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). Our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways. PMID:25415302

  19. Array comparative genomic hybridization-based characterization of genetic alterations in pulmonary neuroendocrine tumors.

    PubMed

    Voortman, Johannes; Lee, Jih-Hsiang; Killian, Jonathan Keith; Suuriniemi, Miia; Wang, Yonghong; Lucchi, Marco; Smith, William I; Meltzer, Paul; Wang, Yisong; Giaccone, Giuseppe

    2010-07-20

    The goal of this study was to characterize and classify pulmonary neuroendocrine tumors based on array comparative genomic hybridization (aCGH). Using aCGH, we performed karyotype analysis of 33 small cell lung cancer (SCLC) tumors, 13 SCLC cell lines, 19 bronchial carcinoids, and 9 gastrointestinal carcinoids. In contrast to the relatively conserved karyotypes of carcinoid tumors, the karyotypes of SCLC tumors and cell lines were highly aberrant. High copy number (CN) gains were detected in SCLC tumors and cell lines in cytogenetic bands encoding JAK2, FGFR1, and MYC family members. In some of those samples, the CN of these genes exceeded 100, suggesting that they could represent driver alterations and potential drug targets in subgroups of SCLC patients. In SCLC tumors, as well as bronchial carcinoids and carcinoids of gastrointestinal origin, recurrent CN alterations were observed in 203 genes, including the RB1 gene and 59 microRNAs of which 51 locate in the DLK1-DIO3 domain. These findings suggest the existence of partially shared CN alterations in these tumor types. In contrast, CN alterations of the TP53 gene and the MYC family members were predominantly observed in SCLC. Furthermore, we demonstrated that the aCGH profile of SCLC cell lines highly resembles that of clinical SCLC specimens. Finally, by analyzing potential drug targets, we provide a genomics-based rationale for targeting the AKT-mTOR and apoptosis pathways in SCLC.

  20. Integrated Genome-Based Studies of Shewanella Ecophysiology

    SciTech Connect

    Andrei L. Osterman, Ph.D.

    2012-12-17

    Integration of bioinformatics and experimental techniques was applied to mapping and characterization of the key components (pathways, enzymes, transporters, regulators) of the core metabolic machinery in Shewanella oneidensis and related species with main focus was on metabolic and regulatory pathways involved in utilization of various carbon and energy sources. Among the main accomplishments reflected in ten joint publications with other participants of Shewanella Federation are: (i) A systems-level reconstruction of carbohydrate utilization pathways in the genus of Shewanella (19 species). This analysis yielded reconstruction of 18 sugar utilization pathways including 10 novel pathway variants and prediction of > 60 novel protein families of enzymes, transporters and regulators involved in these pathways. Selected functional predictions were verified by focused biochemical and genetic experiments. Observed growth phenotypes were consistent with bioinformatic predictions providing strong validation of the technology and (ii) Global genomic reconstruction of transcriptional regulons in 16 Shewanella genomes. The inferred regulatory network includes 82 transcription factors, 8 riboswitches and 6 translational attenuators. Of those, 45 regulons were inferred directly from the genome context analysis, whereas others were propagated from previously characterized regulons in other species. Selected regulatory predictions were experimentally tested. Integration of this analysis with microarray data revealed overall consistency and provided additional layer of interactions between regulons. All the results were captured in the new database RegPrecise, which is a joint development with the LBNL team. A more detailed analysis of the individual subsystems, pathways and regulons in Shewanella spp included bioinfiormatics-based prediction and experimental characterization of: (i) N-Acetylglucosamine catabolic pathway; (ii)Lactate utilization machinery; (iii) Novel Nrt

  1. Genome editing and stem cell therapy pave the path for new treatment of sickle-cell disease.

    PubMed

    Randolph, Mary Katherine; Zhao, Wanke

    2015-01-01

    Sickle-cell disease (SCD), also known as sickle-cell anemia, is a hereditary blood disorder characterized by the presence of abnormal hemoglobin, the oxygen-carrying protein found in red blood cells. This devastating hematologic disease affects millions of children worldwide. Currently the only available cure is an allogenic hematopoietic stem cell transplant (HSCT) which is limited by the scarcity of fully-matched donors. SCD is caused by a single nucleotide mutation in the beta-globin gene. Correction of this genetic defect would provide a cure for the disease. Two recent murine studies have provided proof of principle for such a strategy by correcting the mutation in hematopoietic stem cells (HSC) using genome editing techniques. With transformative advances being made in the genome editing field, effective and precise manipulation of cellular genomes is becoming highly feasible. Genome editing techniques in combination with stem cell therapy should provide a safe and curative treatment of various genetic diseases such as SCD.

  2. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions.

    PubMed

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N; Flood, Beverly E; Bailey, Jake V; Mußmann, Marc

    2016-01-01

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na(+)-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur

  3. Human genome-specific real-time PCR method for sensitive detection and reproducible quantitation of human cells in mice.

    PubMed

    Song, Pengyue; Xie, Zhenhua; Guo, Ling; Wang, Chengmei; Xie, Weidong; Wu, Yaojiong

    2012-12-01

    Xenotransplantation of human cells into immunodeficiency mice has been frequently used to study stem cells in tissue repair and regeneration and cancer cell metastasis. However, a sensitive and reproducible method to quantify cell engraftment lacks. Here, we developed a Real-Time PCR-based method which facilitated consistent detection and quantification of small amounts of human cells distributed in mouse organs after infusion. The principle of the method was to directly detect a humans-specific sequence in the human-murine genomic DNA mixture. In a mouse myocardial infarction model, the Real-Time PCR-based method consistently determined the amounts of human mesenchymal stem cells (hMSCs) engrafted into the heart and other organs 7 days after infusion of as little as 2.5 × 10(5) cells, indicating a high sensitivity, and the amounts of hMSCs detected in mice highly correlated to the numbers of hMSCs transplanted. Importantly, different from previous PCR-based methods, our method produced highly consistent and reproducible results. The reliability of the method was further proven by parallel analyses of DiI-labeled hMSCs in tissue sections and in single cell suspensions of mice. Our data show that the present human genomic DNA-specific primers-based Real-Time PCR method is sensitive and highly reproducible in determining the amount of xenotransplanted human cells in murine tissues.

  4. The genome of the chicken DT40 bursal lymphoma cell line.

    PubMed

    Molnár, János; Póti, Ádám; Pipek, Orsolya; Krzystanek, Marcin; Kanu, Nnennaya; Swanton, Charles; Tusnády, Gábor E; Szallasi, Zoltan; Csabai, István; Szüts, Dávid

    2014-09-15

    The chicken DT40 cell line is a widely used model system in the study of multiple cellular processes due to the efficiency of homologous gene targeting. The cell line was derived from a bursal lymphoma induced by avian leukosis virus infection. In this study we characterized the genome of the cell line using whole genome shotgun sequencing and single nucleotide polymorphism array hybridization. The results indicate that wild-type DT40 has a relatively normal karyotype, except for whole chromosome copy number gains, and no karyotype variability within stocks. In a comparison to two domestic chicken genomes and the Gallus gallus reference genome, we found no unique mutational processes shaping the DT40 genome except for a mild increase in insertion and deletion events, particularly deletions at tandem repeats. We mapped coding sequence mutations that are unique to the DT40 genome; mutations inactivating the PIK3R1 and ATRX genes likely contributed to the oncogenic transformation. In addition to a known avian leukosis virus integration in the MYC gene, we detected further integration sites that are likely to de-regulate gene expression. The new findings support the hypothesis that DT40 is a typical transformed cell line with a relatively intact genome; therefore, it is well-suited to the role of a model system for DNA repair and related processes. The sequence data generated by this study, including a searchable de novo genome assembly and annotated lists of mutated genes, will support future research using this cell line.

  5. G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

    PubMed

    Macaulay, Iain C; Haerty, Wilfried; Kumar, Parveen; Li, Yang I; Hu, Tim Xiaoming; Teng, Mabel J; Goolam, Mubeen; Saurat, Nathalie; Coupland, Paul; Shirley, Lesley M; Smith, Miriam; Van der Aa, Niels; Banerjee, Ruby; Ellis, Peter D; Quail, Michael A; Swerdlow, Harold P; Zernicka-Goetz, Magdalena; Livesey, Frederick J; Ponting, Chris P; Voet, Thierry

    2015-06-01

    The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

  6. A critique of race-based and genomic medicine.

    PubMed

    Meier, Robert J

    2012-03-01

    Now that a composite human genome has been sequenced (HGP), research has accelerated to discover precise genetic bases of several chronic health issues, particularly in the realms of cancer and cardiovascular disease. It is anticipated that in the future it will be possible and cost effective to regularly sequence individual genomes, and thereby produce a DNA profile that potentially can be used to assess the health risks for each person with respect to certain genetically predisposed conditions. Coupled with that enormous diagnostic power, it will then depend upon equally rapid research efforts to develop personalized courses of treatment, including that of pharmaceutical therapy. Initial treatment attempts have been made to match drug efficacy and safety to individuals of assigned or self-identified groups according to their genetic ancestry or presumed race. A prime example is that of BiDil, which was the first drug approved by the US FDA for the explicit treatment of heart patients of African American ancestry. This race-based approach to medicine has been met with justifiable criticism, notably on ethical grounds that have long plagued historical applications and misuses of human race classification, and also on questionable science. This paper will assess race-based medical research and practice in light of a more thorough understanding of human genetic variability. Additional concerns will be expressed with regard to the rapidly developing area of pharmacogenomics, promoted to be the future of personalized medicine. Genomic epidemiology will be discussed with several examples of on-going research that hopefully will provide a solid scientific grounding for personalized medicine to build upon.

  7. Single-cell Genomics Unveils Critical Regulators of Th17 cell Pathogenicity

    PubMed Central

    Gaublomme, Jellert T.; Yosef, Nir; Lee, Youjin; Gertner, Rona S.; Yang, Li V.; Wu, Chuan; Pandolfi, Pier Paolo; Mak, Tak; Satija, Rahul; Shalek, Alex K.; Kuchroo, Vijay K.; Park, Hongkun; Regev, Aviv

    2015-01-01

    SUMMARY Extensive cellular heterogeneity exists within specific immune-cell subtypes classified as a single lineage, but its molecular underpinnings are rarely characterized at a genomic scale. Here, we use single-cell RNA-seq to investigate the molecular mechanisms governing heterogeneity and pathogenicity of Th17 cells isolated from the central nervous system (CNS) and lymph nodes (LN) at the peak of autoimmune encephalomyelitis (EAE) or differentiated in vitro under either pathogenic or non-pathogenic polarization conditions. Computational analysis relates a spectrum of cellular states in vivo to in vitro differentiated Th17 cells, and unveils genes governing pathogenicity and disease susceptibility. Using knockout mice, we validate four new genes: Gpr65, Plzp, Toso and Cd5l (in a companion paper). Cellular heterogeneity thus informs Th17 function in autoimmunity, and can identify targets for selective suppression of pathogenic Th17 cells while potentially sparing non-pathogenic tissue-protective ones. PMID:26607794

  8. Zscan4 regulates telomere elongation and genomic stability in ES cells

    PubMed Central

    Zalzman, Michal; Falco, Geppino; Sharova, Lioudmila V.; Nishiyama, Akira; Thomas, Marshall; Lee, Sung-Lim; Stagg, Carole A.; Hoang, Hien G.; Yang, Hsih-Te; Indig, Fred E.; Wersto, Robert P.; Ko, Minoru S. H.

    2010-01-01

    Exceptional genomic stability is one of the hallmarks of mouse embryonic stem (ES) cells. However, the genes contributing to this stability remain obscure. We previously identified Zscan4 as a specific marker for 2-cell embryo and ES cells. Here we show that Zscan4 is involved in telomere maintenance and long-term-genomic stability in ES cells. Only 5% of ES cells express Zscan4 at a given time, but nearly all ES cells activate Zscan4 at least once within nine passages. The transient Zscan4-positive state is associated with rapid telomere extension by telomere recombination and upregulation of meiosis-specific homologous recombination genes, which encode proteins that are colocalized with ZSCAN4 on telomeres. Furthermore, Zscan4 knockdown shortens telomeres, increases karyotype abnormalities and spontaneous sister chromatid exchange, and slows down cell proliferation until reaching crisis by eight passages. Together, our data reveal a unique mode of genome maintenance in ES cells. PMID:20336070

  9. Ectopic Expression of Testis Germ Cell Proteins in Cancer and Its Potential Role in Genomic Instability

    PubMed Central

    Nielsen, Aaraby Yoheswaran; Gjerstorff, Morten Frier

    2016-01-01

    Genomic instability is a hallmark of human cancer and an enabling factor for the genetic alterations that drive cancer development. The processes involved in genomic instability resemble those of meiosis, where genetic material is interchanged between homologous chromosomes. In most types of human cancer, epigenetic changes, including hypomethylation of gene promoters, lead to the ectopic expression of a large number of proteins normally restricted to the germ cells of the testis. Due to the similarities between meiosis and genomic instability, it has been proposed that activation of meiotic programs may drive genomic instability in cancer cells. Some germ cell proteins with ectopic expression in cancer cells indeed seem to promote genomic instability, while others reduce polyploidy and maintain mitotic fidelity. Furthermore, oncogenic germ cell proteins may indirectly contribute to genomic instability through induction of replication stress, similar to classic oncogenes. Thus, current evidence suggests that testis germ cell proteins are implicated in cancer development by regulating genomic instability during tumorigenesis, and these proteins therefore represent promising targets for novel therapeutic strategies. PMID:27275820

  10. Automating Genomic Data Mining via a Sequence-based Matrix Format and Associative Rule Set

    PubMed Central

    Wren, Jonathan D; Johnson, David; Gruenwald, Le

    2005-01-01

    There is an enormous amount of information encoded in each genome – enough to create living, responsive and adaptive organisms. Raw sequence data alone is not enough to understand function, mechanisms or interactions. Changes in a single base pair can lead to disease, such as sickle-cell anemia, while some large megabase deletions have no apparent phenotypic effect. Genomic features are varied in their data types and annotation of these features is spread across multiple databases. Herein, we develop a method to automate exploration of genomes by iteratively exploring sequence data for correlations and building upon them. First, to integrate and compare different annotation sources, a sequence matrix (SM) is developed to contain position-dependant information. Second, a classification tree is developed for matrix row types, specifying how each data type is to be treated with respect to other data types for analysis purposes. Third, correlative analyses are developed to analyze features of each matrix row in terms of the other rows, guided by the classification tree as to which analyses are appropriate. A prototype was developed and successful in detecting coinciding genomic features among genes, exons, repetitive elements and CpG islands. PMID:16026599

  11. Genome engineering of isogenic human ES cells to model autism disorders

    PubMed Central

    Martinez, Refugio A.; Stein, Jason L.; Krostag, Anne-Rachel F.; Nelson, Angelique M.; Marken, John S.; Menon, Vilas; May, Ryan C.; Yao, Zizhen; Kaykas, Ajamete; Geschwind, Daniel H.; Grimley, Joshua S.

    2015-01-01

    Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity. PMID:25765640

  12. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    SciTech Connect

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. )

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  13. A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response. | Office of Cancer Genomics

    Cancer.gov

    Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis.

  14. GSP: A web-based platform for designing genome-specific primers in polyploids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The sequences among subgenomes in a polyploid species have high similarity. This makes difficult to design genome-specific primers for sequence analysis. We present a web-based platform named GSP for designing genome-specific primers to distinguish subgenome sequences in the polyploid genome backgr...

  15. BeetleBase in 2010: Revisions to Provide Comprehensive Genomic Information for Tribolium castaneum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BeetleBase (http://www.beetlebase.org) has been updated to provide more comprehensive genomic information for the red flour beetle Tribolium castaneum. The database contains genomic sequence scaffolds mapped to 10 linkage groups (genome assembly release Tcas_3.0), genetic linkage maps, the official ...

  16. Ascaris phylogeny based on multiple whole mtDNA genomes.

    PubMed

    Nejsum, Peter; Hawash, Mohamed B F; Betson, Martha; Stothard, J Russell; Gasser, Robin B; Andersen, Lee O

    2017-03-01

    Ascaris lumbricoides and A. suum are two parasitic nematodes infecting humans and pigs, respectively. There has been considerable debate as to whether Ascaris in the two hosts should be considered a single or two separate species. Previous studies identified at least three major clusters (A, B and C) of human and pig Ascaris based on partial cox1 sequences. In the present study, we selected major haplotypes from these different clusters to characterize their whole mitochondrial genomes for phylogenetic analysis. We also undertook coalescent simulations to investigate the evolutionary history of the different Ascaris haplotypes. The topology of the phylogenetic tree based on complete mitochondrial genomic sequences was found to be similar to partial cox1 sequencing, but the support at internal nodes was higher in the former. Coalescent simulations suggested the presence of at least two divergence events: the first one occurring early in the Neolithic period which resulted in a differentiated population of Ascaris in pigs (cluster C), the second occurring more recently (~900 generations ago), resulting in clusters A and B which might have been spread worldwide by human activities.

  17. MLL leukemia induction by genome editing of human CD34+ hematopoietic cells

    PubMed Central

    Buechele, Corina; Breese, Erin H.; Schneidawind, Dominik; Lin, Chiou-Hong; Jeong, Johan; Duque-Afonso, Jesus; Wong, Stephen H. K.; Smith, Kevin S.; Negrin, Robert S.; Porteus, Matthew

    2015-01-01

    Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease–mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia. PMID:26311362

  18. MLL leukemia induction by genome editing of human CD34+ hematopoietic cells.

    PubMed

    Buechele, Corina; Breese, Erin H; Schneidawind, Dominik; Lin, Chiou-Hong; Jeong, Johan; Duque-Afonso, Jesus; Wong, Stephen H K; Smith, Kevin S; Negrin, Robert S; Porteus, Matthew; Cleary, Michael L

    2015-10-01

    Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease-mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.

  19. Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients.

    PubMed

    Ruiz, Carmen; Li, Julia; Luttgen, Madelyn S; Kolatkar, Anand; Kendall, Jude T; Flores, Edna; Topp, Zheng; Samlowski, Wolfram E; McClay, Edward; Bethel, Kelly; Ferrone, Soldano; Hicks, James; Kuhn, Peter

    2015-01-09

    Purpose. Circulating melanoma cells (CMCs) constitute a potentially important representation of time-resolved tumor biology in patients. To date, genomic characterization of CMCs has been limited due to the lack of a robust methodology capable of identifying them in a format suitable for downstream characterization. Here, we have developed a methodology to detect intact CMCs that enables phenotypic, morphometric and genomic analysis at the single cell level. Experimental design. Blood samples from 40 metastatic melanoma patients and 10 normal blood donors were prospectively collected. A panel of 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAbs) was used to immunocytochemically label CMCs. Detection was performed by automated digital fluorescence microscopy and multi-parametric computational analysis. Individual CMCs were captured by micromanipulation for whole genome amplification and copy number variation (CNV) analysis. Results. Based on CSPG4 expression and nuclear size, 1-250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5-371.5 CMCs ml(-1)). Morphometric analysis revealed that CMCs have a broad spectrum of morphologies and sizes but exhibit a relatively homogeneous nuclear size that was on average 1.5-fold larger than that of surrounding PBMCs. CNV analysis of single CMCs identified deletions of CDKN2A and PTEN, and amplification(s) of TERT, BRAF, KRAS and MDM2. Furthermore, novel chromosomal amplifications in chr12, 17 and 19 were also found. Conclusions. Our findings show that CSPG4 expressing CMCs can be found in the majority of advanced melanoma patients. High content analysis of this cell population may contribute to the design of effective personalized therapies in patients with melanoma.

  20. Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients

    NASA Astrophysics Data System (ADS)

    Ruiz, Carmen; Li, Julia; Luttgen, Madelyn S.; Kolatkar, Anand; Kendall, Jude T.; Flores, Edna; Topp, Zheng; Samlowski, Wolfram E.; McClay, Edward; Bethel, Kelly; Ferrone, Soldano; Hicks, James; Kuhn, Peter

    2015-02-01

    Purpose. Circulating melanoma cells (CMCs) constitute a potentially important representation of time-resolved tumor biology in patients. To date, genomic characterization of CMCs has been limited due to the lack of a robust methodology capable of identifying them in a format suitable for downstream characterization. Here, we have developed a methodology to detect intact CMCs that enables phenotypic, morphometric and genomic analysis at the single cell level. Experimental design. Blood samples from 40 metastatic melanoma patients and 10 normal blood donors were prospectively collected. A panel of 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAbs) was used to immunocytochemically label CMCs. Detection was performed by automated digital fluorescence microscopy and multi-parametric computational analysis. Individual CMCs were captured by micromanipulation for whole genome amplification and copy number variation (CNV) analysis. Results. Based on CSPG4 expression and nuclear size, 1-250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5-371.5 CMCs ml-1). Morphometric analysis revealed that CMCs have a broad spectrum of morphologies and sizes but exhibit a relatively homogeneous nuclear size that was on average 1.5-fold larger than that of surrounding PBMCs. CNV analysis of single CMCs identified deletions of CDKN2A and PTEN, and amplification(s) of TERT, BRAF, KRAS and MDM2. Furthermore, novel chromosomal amplifications in chr12, 17 and 19 were also found. Conclusions. Our findings show that CSPG4 expressing CMCs can be found in the majority of advanced melanoma patients. High content analysis of this cell population may contribute to the design of effective personalized therapies in patients with melanoma.

  1. Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome

    PubMed Central

    Gu, Junchen; Stevens, Michael; Xing, Xiaoyun; Li, Daofeng; Zhang, Bo; Payton, Jacqueline E.; Oltz, Eugene M.; Jarvis, James N.; Jiang, Kaiyu; Cicero, Theodore; Costello, Joseph F.; Wang, Ting

    2016-01-01

    DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types. PMID:26888867

  2. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.

    PubMed

    Zhu, Xiaoxiao; Xu, Yajie; Yu, Shanshan; Lu, Lu; Ding, Mingqin; Cheng, Jing; Song, Guoxu; Gao, Xing; Yao, Liangming; Fan, Dongdong; Meng, Shu; Zhang, Xuewen; Hu, Shengdi; Tian, Yong

    2014-09-19

    The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.

  3. Inferences of drug responses in cancer cells from cancer genomic features and compound chemical and therapeutic properties

    NASA Astrophysics Data System (ADS)

    Wang, Yongcui; Fang, Jianwen; Chen, Shilong

    2016-09-01

    Accurately predicting the response of a cancer patient to a therapeutic agent is a core goal of precision medicine. Existing approaches were mainly relied primarily on genomic alterations in cancer cells that have been treated with different drugs. Here we focus on predicting drug response based on integration of the heterogeneously pharmacogenomics data from both cell and drug sides. Through a systematical approach, named as PDRCC (Predict Drug Response in Cancer Cells), the cancer genomic alterations and compound chemical and therapeutic properties were incorporated to determine the chemotherapeutic response in cancer patients. Using the Cancer Cell Line Encyclopedia (CCLE) study as the benchmark dataset, all pharmacogenomics data exhibited their roles in inferring the relationships between cancer cells and drugs. When integrating both genomic resources and compound information, the prediction coverage was significantly increased. The validity of PDRCC was also supported by its effective in uncovering the unknown cell-drug associations with database and literature evidences. It set the stage for clinical testing of novel therapeutic strategies, such as the sensitive association between cancer cell ‘A549_LUNG’ and compound ‘Topotecan’. In conclusion, PDRCC offers the possibility for faster, safer, and cheaper the development of novel anti-cancer therapeutics in the early-stage clinical trails.

  4. Inferences of drug responses in cancer cells from cancer genomic features and compound chemical and therapeutic properties

    PubMed Central

    Wang, Yongcui; Fang, Jianwen; Chen, Shilong

    2016-01-01

    Accurately predicting the response of a cancer patient to a therapeutic agent is a core goal of precision medicine. Existing approaches were mainly relied primarily on genomic alterations in cancer cells that have been treated with different drugs. Here we focus on predicting drug response based on integration of the heterogeneously pharmacogenomics data from both cell and drug sides. Through a systematical approach, named as PDRCC (Predict Drug Response in Cancer Cells), the cancer genomic alterations and compound chemical and therapeutic properties were incorporated to determine the chemotherapeutic response in cancer patients. Using the Cancer Cell Line Encyclopedia (CCLE) study as the benchmark dataset, all pharmacogenomics data exhibited their roles in inferring the relationships between cancer cells and drugs. When integrating both genomic resources and compound information, the prediction coverage was significantly increased. The validity of PDRCC was also supported by its effective in uncovering the unknown cell-drug associations with database and literature evidences. It set the stage for clinical testing of novel therapeutic strategies, such as the sensitive association between cancer cell ‘A549_LUNG’ and compound ‘Topotecan’. In conclusion, PDRCC offers the possibility for faster, safer, and cheaper the development of novel anti-cancer therapeutics in the early-stage clinical trails. PMID:27645580

  5. Accurate whole genome sequencing and haplotyping from10-20 human cells

    PubMed Central

    Peters, Brock A.; Kermani, Bahram G.; Sparks, Andrew B.; Alferov, Oleg; Hong, Peter; Alexeev, Andrei; Jiang, Yuan; Dahl, Fredrik; Tang, Y. Tom; Haas, Juergen; Robasky, Kimberly; Zaranek, Alexander Wait; Lee, Je-Hyuk; Ball, Madeleine Price; Peterson, Joseph E.; Perazich, Helena; Yeung, George; Liu, Jia; Chen, Linsu; Kennemer, Michael I.; Pothuraju, Kaliprasad; Konvicka, Karel; Tsoupko-Sitnikov, Mike; Pant, Krishna P.; Ebert, Jessica C.; Nilsen, Geoffrey B.; Baccash, Jonathan; Halpern, Aaron L.; Church, George M.; Drmanac, Radoje

    2012-01-01

    Recent advances in whole genome sequencing have brought the vision of personal genomics and genomic medicine closer to reality. However, current methods lack clinical accuracy and the ability to describe the context (haplotypes) in which genome variants co-occur in a cost-effective manner. Here we describe a low-cost DNA sequencing and haplotyping process, Long Fragment Read (LFR) technology, similar to sequencing long single DNA molecules without cloning or separation of metaphase chromosomes. In this study, ten LFR libraries were made using only ~100 pg of human DNA per sample. Up to 97% of the heterozygous single nucleotide variants (SNVs) were assembled into long haplotype contigs. Removal of false positive SNVs not phased by multiple LFR haplotypes resulted in a final genome error rate of 1 in 10 Mb. Cost-effective and accurate genome sequencing and haplotyping from 10-20 human cells, as demonstrated here, will enable comprehensive genetic studies and diverse clinical applications. PMID:22785314

  6. Functional genomics in the study of yeast cell polarity: moving in the right direction.

    PubMed

    Styles, Erin; Youn, Ji-Young; Mattiazzi Usaj, Mojca; Andrews, Brenda

    2013-01-01

    The budding yeast Saccharomyces cerevisiae has been used extensively for the study of cell polarity, owing to both its experimental tractability and the high conservation of cell polarity and other basic biological processes among eukaryotes. The budding yeast has also served as a pioneer model organism for virtually all genome-scale approaches, including functional genomics, which aims to define gene function and biological pathways systematically through the analysis of high-throughput experimental data. Here, we outline the contributions of functional genomics and high-throughput methodologies to the study of cell polarity in the budding yeast. We integrate data from published genetic screens that use a variety of functional genomics approaches to query different aspects of polarity. Our integrated dataset is enriched for polarity processes, as well as some processes that are not intrinsically linked to cell polarity, and may provide new areas for future study.

  7. On the road to synthetic life: the minimal cell and genome-scale engineering.

    PubMed

    Juhas, Mario

    2016-01-01

    Synthetic biology employs rational engineering principles to build biological systems from the libraries of standard, well characterized biological parts. Biological systems designed and built by synthetic biologists fulfill a plethora of useful purposes, ranging from better healthcare and energy production to biomanufacturing. Recent advancements in the synthesis, assembly and "booting-up" of synthetic genomes and in low and high-throughput genome engineering have paved the way for engineering on the genome-wide scale. One of the key goals of genome engineering is the construction of minimal genomes consisting solely of essential genes (genes indispensable for survival of living organisms). Besides serving as a toolbox to understand the universal principles of life, the cell encoded by minimal genome could be used to build a stringently controlled "cell factory" with a desired phenotype. This review provides an update on recent advances in the genome-scale engineering with particular emphasis on the engineering of minimal genomes. Furthermore, it presents an ongoing discussion to the scientific community for better suitability of minimal or robust cells for industrial applications.

  8. Mutation of Breast Cancer Cell Genomic DNA by APOBEC3B

    DTIC Science & Technology

    2012-09-01

    Award Number: W81XWH-11-1-0014 TITLE: Mutation of Breast Cancer Cell Genomic DNA by APOBEC3B...September 2012 2. REPORT TYPE ANNUAL SUMMARY 3. DATES COVERED 01 Sep 2012 – 31 Aug 2012 4. TITLE AND SUBTITLE Mutation of Breast Cancer Cell Genomic...Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Many breast cancers have somatic mutation spectra dominated by C-to

  9. Complete genome sequence and cell structure of Limnochorda pilosa, a Gram-negative spore former within the phylum Firmicutes.

    PubMed

    Watanabe, Miho; Kojima, Hisaya; Fukui, Manabu

    2016-01-06

    Limnochorda pilosa is a pleomorphic facultative anaerobe and the sole species in the class Limnochordia, which has tentatively been placed in the phylum Firmicutes. In the present study, the complete genome sequence of L. pilosa HC45T was obtained and analyzed. The genome size was 3.82 Mbp and the G+C content was 69.73%. Phylogenetic analyses based on the 30S-50S ribosomal proteins and 23S rRNA gene consistently indicated that L. pilosa is phylogenetically isolated from the other members of the phylum Firmicutes. Ultrastructural observation revealed that L. pilosa possesses a Gram-negative-type cell wall and the capacity to form endospores. Accordingly, the L. pilosa genome has characteristics that are specific to Gram-negative bacteria and contains many genes that are involved in sporulation. On the other hand, several sporulation genes were absent in L. pilosa genome although they have been regarded as essential for endospore-forming system of the phylum Firmicutes. The gyrB gene of L. pilosa possesses an intein sequence. The genome has a high percentage of GTG start codons and lacks several conserved genes related to cell division.

  10. Genome-scale constraint-based modeling of Geobacter metallireducens

    PubMed Central

    Sun, Jun; Sayyar, Bahareh; Butler, Jessica E; Pharkya, Priti; Fahland, Tom R; Famili, Iman; Schilling, Christophe H; Lovley, Derek R; Mahadevan, Radhakrishnan

    2009-01-01

    Background Geobacter metallireducens was the first organism that can be grown in pure culture to completely oxidize organic compounds with Fe(III) oxide serving as electron acceptor. Geobacter species, including G. sulfurreducens and G. metallireducens, are used for bioremediation and electricity generation from waste organic matter and renewable biomass. The constraint-based modeling approach enables the development of genome-scale in silico models that can predict the behavior of complex biological systems and their responses to the environments. Such a modeling approach was applied to provide physiological and ecological insights on the metabolism of G. metallireducens. Results The genome-scale metabolic model of G. metallireducens was constructed to include 747 genes and 697 reactions. Compared to the G. sulfurreducens model, the G. metallireducens metabolic model contains 118 unique reactions that reflect many of G. metallireducens' specific metabolic capabilities. Detailed examination of the G. metallireducens model suggests that its central metabolism contains several energy-inefficient reactions that are not present in the G. sulfurreducens model. Experimental biomass yield of G. metallireducens growing on pyruvate was lower than the predicted optimal biomass yield. Microarray data of G. metallireducens growing with benzoate and acetate indicated that genes encoding these energy-inefficient reactions were up-regulated by benzoate. These results suggested that the energy-inefficient reactions were likely turned off during G. metallireducens growth with acetate for optimal biomass yield, but were up-regulated during growth with complex electron donors such as benzoate for rapid energy generation. Furthermore, several computational modeling approaches were applied to accelerate G. metallireducens research. For example, growth of G. metallireducens with different electron donors and electron acceptors were studied using the genome-scale metabolic model, which

  11. FlyBase: introduction of the Drosophila melanogaster Release 6 reference genome assembly and large-scale migration of genome annotations

    PubMed Central

    dos Santos, Gilberto; Schroeder, Andrew J.; Goodman, Joshua L.; Strelets, Victor B.; Crosby, Madeline A.; Thurmond, Jim; Emmert, David B.; Gelbart, William M.

    2015-01-01

    Release 6, the latest reference genome assembly of the fruit fly Drosophila melanogaster, was released by the Berkeley Drosophila Genome Project in 2014; it replaces their previous Release 5 genome assembly, which had been the reference genome assembly for over 7 years. With the enormous amount of information now attached to the D. melanogaster genome in public repositories and individual laboratories, the replacement of the previous assembly by the new one is a major event requiring careful migration of annotations and genome-anchored data to the new, improved assembly. In this report, we describe the attributes of the new Release 6 reference genome assembly, the migration of FlyBase genome annotations to this new assembly, how genome features on this new assembly can be viewed in FlyBase (http://flybase.org) and how users can convert coordinates for their own data to the corresponding Release 6 coordinates. PMID:25398896

  12. FlyBase: introduction of the Drosophila melanogaster Release 6 reference genome assembly and large-scale migration of genome annotations.

    PubMed

    dos Santos, Gilberto; Schroeder, Andrew J; Goodman, Joshua L; Strelets, Victor B; Crosby, Madeline A; Thurmond, Jim; Emmert, David B; Gelbart, William M

    2015-01-01

    Release 6, the latest reference genome assembly of the fruit fly Drosophila melanogaster, was released by the Berkeley Drosophila Genome Project in 2014; it replaces their previous Release 5 genome assembly, which had been the reference genome assembly for over 7 years. With the enormous amount of information now attached to the D. melanogaster genome in public repositories and individual laboratories, the replacement of the previous assembly by the new one is a major event requiring careful migration of annotations and genome-anchored data to the new, improved assembly. In this report, we describe the attributes of the new Release 6 reference genome assembly, the migration of FlyBase genome annotations to this new assembly, how genome features on this new assembly can be viewed in FlyBase (http://flybase.org) and how users can convert coordinates for their own data to the corresponding Release 6 coordinates.

  13. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis.

    PubMed

    Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum; Delay, Catherine E; Yang, Zhang; Niola, Francesco; Duda, Katarzyna; Ober, Elke A; Clausen, Henrik; Wandall, Hans H; Hansen, Steen H; Bennett, Eric P; Frödin, Morten

    2017-03-01

    This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.

  14. Identification of Ohnolog Genes Originating from Whole Genome Duplication in Early Vertebrates, Based on Synteny Comparison across Multiple Genomes.

    PubMed

    Singh, Param Priya; Arora, Jatin; Isambert, Hervé

    2015-07-01

    Whole genome duplications (WGD) have now been firmly established in all major eukaryotic kingdoms. In particular, all vertebrates descend from two rounds of WGDs, that occurred in their jawless ancestor some 500 MY ago. Paralogs retained from WGD, also coined 'ohnologs' after Susumu Ohno, have been shown to be typically associated with development, signaling and gene regulation. Ohnologs, which amount to about 20 to 35% of genes in the human genome, have also been shown to be prone to dominant deleterious mutations and frequently implicated in cancer and genetic diseases. Hence, identifying ohnologs is central to better understand the evolution of vertebrates and their susceptibility to genetic diseases. Early computational analyses to identify vertebrate ohnologs relied on content-based synteny comparisons between the human genome and a single invertebrate outgroup genome or within the human genome itself. These approaches are thus limited by lineage specific rearrangements in individual genomes. We report, in this study, the identification of vertebrate ohnologs based on the quantitative assessment and integration of synteny conservation between six amniote vertebrates and six invertebrate outgroups. Such a synteny comparison across multiple genomes is shown to enhance the statistical power of ohnolog identification in vertebrates compared to earlier approaches, by overcoming lineage specific genome rearrangements. Ohnolog gene families can be browsed and downloaded for three statistical confidence levels or recompiled for specific, user-defined, significance criteria at http://ohnologs.curie.fr/. In the light of the importance of WGD on the genetic makeup of vertebrates, our analysis provides a useful resource for researchers interested in gaining further insights on vertebrate evolution and genetic diseases.

  15. Identification of the glucosyltransferase that converts hydroxymethyluracil to base J in the trypanosomatid genome.

    PubMed

    Bullard, Whitney; Lopes da Rosa-Spiegler, Jessica; Liu, Shuo; Wang, Yinsheng; Sabatini, Robert

    2014-07-18

    O-linked glucosylation of thymine in DNA (base J) is an important regulatory epigenetic mark in trypanosomatids. β-d-glucopyranosyloxymethyluracil (base J) synthesis is initiated by the JBP1/2 enzymes that hydroxylate thymine, forming 5-hydroxymethyluracil (hmU). hmU is then glucosylated by a previously unknown glucosyltransferase. A recent computational screen identified a possible candidate for the base J-associated glucosyltransferase (JGT) in trypanosomatid genomes. We demonstrate that recombinant JGT utilizes uridine diphosphoglucose to transfer glucose to hmU in the context of dsDNA. Mutation of conserved residues typically involved in glucosyltransferase catalysis impairs DNA glucosylation in vitro. The deletion of both alleles of JGT from the genome of Trypanosoma brucei generates a cell line that completely lacks base J. Reintroduction of JGT in the JGT KO restores J synthesis. Ablation of JGT mRNA levels by RNAi leads to the sequential reduction in base J and increased levels of hmU that dissipate rapidly. The analysis of JGT function confirms the two-step J synthesis model and demonstrates that JGT is the only glucosyltransferase enzyme required for the second step of the pathway. Similar to the activity of the related Ten-Eleven Translocation (TET) family of dioxygenases on 5mC, our studies also suggest the ability of the base J-binding protein enzymes to catalyze iterative oxidation of thymine in trypanosome DNA. Here we discuss the regulation of hmU and base J formation in the trypanosome genome by JGT and base J-binding protein.

  16. Manipulation of a Single Circulating Tumor Cell Using Visualization of Hydrogel Encapsulation toward Single-Cell Whole-Genome Amplification.

    PubMed

    Yoshino, Tomoko; Tanaka, Tsuyoshi; Nakamura, Seita; Negishi, Ryo; Hosokawa, Masahito; Matsunaga, Tadashi

    2016-07-19

    Genetic characterization of circulating tumor cells (CTCs) could guide the choice of therapies for individual patients and also facilitate the development of new drugs. We previously developed a CTC recovery system using a microcavity array, which demonstrated highly efficient CTC recovery based on differences in cell size and deformability. However, the CTC recovery system lacked an efficient cell manipulation tool suitable for subsequent genetic analysis. Here, we resolve this issue and present a simple and rapid manipulation method for single CTCs using a photopolymerized hydrogel, polyethylene glycol diacrylate (PEGDA), which is useful for subsequent genetic analysis. First, PEGDA was introduced into the cells entrapped on the microcavity array. Then, excitation light was projected onto the target single cells for encapsulation of each CTC by confocal laser-scanning microscopy. The encapsulated single CTCs could be visualized by the naked eye and easily handled with tweezers. The single CTCs were only partially encapsulated on the PEGDA hydrogel, which allowed for sufficient whole-genome amplification and accurate genotyping. Our proposed methodology is a valuable tool for the rapid and simple manipulation of single CTCs and is expected to become widely utilized for analyses of mammalian cells and microorganisms in addition to CTCs.

  17. Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines

    PubMed Central

    Nagel, Stefan; Eberth, Sonja; Pommerenke, Claudia; Dirks, Wilhelm G.; Geffers, Robert; Kalavalapalli, Srilaxmi; Kaufmann, Maren; Meyer, Corrina; Faehnrich, Silke; Chen, Suning; Drexler, Hans G.; MacLeod, Roderick A. F.

    2015-01-01

    Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2–10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings

  18. Draft Genome Sequence of Acholeplasma laidlawii, a Common Contaminant of Cell Cultures.

    PubMed

    Siqueira, Franciele Maboni; Cibulski, Samuel Paulo; Teixeira, Thais Fumaco; Mayer, Fabiana Quoos; Roehe, Paulo Michel

    2017-02-02

    Mollicutes are important cell culture contaminants which may eventually affect the results of biological assays or affect their interpretation. Acholeplasma laidlawii is one of the most frequent contaminants of cell cultures. Here, we report the complete genome sequence of A. laidlawii strain MDBK/IPV, recovered from Madin-Darby bovine kidney (MDBK) cells.

  19. Gene Expression Correlates with the Number of Herpes Viral Genomes Initiating Infection in Single Cells

    PubMed Central

    Cohen, Efrat M.

    2016-01-01

    Viral gene expression varies significantly among genetically identical cells. The sources of these variations are not well understood and have been suggested to involve both deterministic host differences and stochastic viral host interactions. For herpesviruses, only a limited number of incoming viral genomes initiate expression and replication in each infected cell. To elucidate the effect of this limited number of productively infecting genomes on viral gene expression in single cells, we constructed a set of fluorescence-expressing genetically tagged herpes recombinants. The number of different barcodes originating from a single cell is a good representative of the number of incoming viral genomes replicating (NOIVGR) in that cell. We identified a positive correlation between the NOIVGR and viral gene expression, as measured by the fluorescent protein expressed from the viral genome. This correlation was identified in three distinct cell-types, although the average NOIVGR per cell differed among these cell-types. Among clonal single cells, high housekeeping gene expression levels are not supportive of high viral gene expression, suggesting specific host determinants effecting viral infection. We developed a model to predict NOIVGR from cellular parameters, which supports the notion that viral gene expression is tightly linked to the NOIVGR in single-cells. Our results support the hypothesis that the stochastic nature of viral infection and host cell determinants contribute together to the variability observed among infected cells. PMID:27923068

  20. Draft Genome Sequence of Acholeplasma laidlawii, a Common Contaminant of Cell Cultures

    PubMed Central

    Siqueira, Franciele Maboni; Cibulski, Samuel Paulo; Teixeira, Thais Fumaco; Mayer, Fabiana Quoos

    2017-01-01

    ABSTRACT Mollicutes are important cell culture contaminants which may eventually affect the results of biological assays or affect their interpretation. Acholeplasma laidlawii is one of the most frequent contaminants of cell cultures. Here, we report the complete genome sequence of A. laidlawii strain MDBK/IPV, recovered from Madin-Darby bovine kidney (MDBK) cells. PMID:28153907

  1. Parallel Single Cancer Cell Whole Genome Amplification Using Button-Valve Assisted Mixing in Nanoliter Chambers

    PubMed Central

    Yang, Yoonsun; Swennenhuis, Joost F.; Rho, Hoon Suk; Le Gac, Séverine; Terstappen, Leon W. M. M.

    2014-01-01

    The heterogeneity of tumor cells and their alteration during the course of the disease urges the need for real time characterization of individual tumor cells to improve the assessment of treatment options. New generations of therapies are frequently associated with specific genetic alterations driving the need to determine the genetic makeup of tumor cells. Here, we present a microfluidic device for parallel single cell whole genome amplification (pscWGA) to obtain enough copies of a single cell genome to probe for the presence of treatment targets and the frequency of its occurrence among the tumor cells. Individual cells were first captured and loaded into eight parallel amplification units. Next, cells were lysed on a chip and their DNA amplified through successive introduction of dedicated reagents while mixing actively with the help of integrated button-valves. The reaction chamber volume for scWGA 23.85 nl, and starting from 6–7 pg DNA contained in a single cell, around 8 ng of DNA was obtained after WGA, representing over 1000-fold amplification. The amplified products from individual breast cancer cells were collected from the device to either directly investigate the amplification of specific genes by qPCR or for re-amplification of the DNA to obtain sufficient material for whole genome sequencing. Our pscWGA device provides sufficient DNA from individual cells for their genetic characterization, and will undoubtedly allow for automated sample preparation for single cancer cell genomic characterization. PMID:25233459

  2. The iCRISPR platform for rapid genome editing in human pluripotent stem cells.

    PubMed

    Zhu, Zengrong; González, Federico; Huangfu, Danwei

    2014-01-01

    Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. Here, we first describe the establishment of the iCRISPR platform through TALEN-mediated targeting of inducible Cas9 expression cassettes into the AAVS1 locus. Next, we provide a series of technical procedures for using iCRISPR to achieve one-step knockout of one or multiple gene(s), "scarless" introduction of precise nucleotide alterations, as well as inducible knockout during hPSC differentiation. We present an optimized workflow, as well as guidelines for the selection of CRISPR targeting sequences and the design of single-stranded DNA (ssDNA) homology-directed DNA repair templates for the introduction of specific nucleotide alterations. We have successfully used these protocols in four different hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic modifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease.

  3. From Agrobacterium to viral vectors: genome modification of plant cells by rare cutting restriction enzymes.

    PubMed

    Marton, Ira; Honig, Arik; Omid, Ayelet; De Costa, Noam; Marhevka, Elena; Cohen, Barry; Zuker, Amir; Vainstein, Alexander

    2013-01-01

    Researchers and biotechnologists require methods to accurately modify the genome of higher eukaryotic cells. Such modifications include, but are not limited to, site-specific mutagenesis, site-specific insertion of foreign DNA, and replacement and deletion of native sequences. Accurate genome modifications in plant species have been rather limited, with only a handful of plant species and genes being modified through the use of early genome-editing techniques. The development of rare-cutting restriction enzymes as a tool for the induction of site-specific genomic double-strand breaks and their introduction as a reliable tool for genome modification in animals, animal cells and human cell lines have paved the way for the adaptation of rare-cutting restriction enzymes to genome editing in plant cells. Indeed, the number of plant species and genes which have been successfully edited using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered homing endonucleases is on the rise. In our review, we discuss the basics of rare-cutting restriction enzyme-mediated genome-editing technology with an emphasis on its application in plant species.

  4. CRISPR/Cas9-mediated genome engineering of CHO cell factories: Application and perspectives.

    PubMed

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E; Faustrup Kildegaard, Helene

    2015-07-01

    Chinese hamster ovary (CHO) cells are the most widely used production host for therapeutic proteins. With the recent emergence of CHO genome sequences, CHO cell line engineering has taken on a new aspect through targeted genome editing. The bacterial clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid, easy and efficient engineering of mammalian genomes. It has a wide range of applications from modification of individual genes to genome-wide screening or regulation of genes. Facile genome editing using CRISPR/Cas9 empowers researchers in the CHO community to elucidate the mechanistic basis behind high level production of proteins and product quality attributes of interest. In this review, we describe the basis of CRISPR/Cas9-mediated genome editing and its application for development of next generation CHO cell factories while highlighting both future perspectives and challenges. As one of the main drivers for the CHO systems biology era, genome engineering with CRISPR/Cas9 will pave the way for rational design of CHO cell factories.

  5. Comparisons of Shewanella strains based on genome annotations, modeling and experiments

    SciTech Connect

    Ong, Wai Kit; Vu, Trang; Lovendahl, Klaus N.; Llull, Jenna; Serres, Margaret; Romine, Margaret F.; Reed, Jennifer L.

    2014-01-01

    Shewanella is a genus of facultatively anaerobic, Gram-negative bacteria that have highly adaptable metabolism which allows them to thrive in diverse environments. This quality makes them attractive target bacteria for research in bioremediation and microbial fuel cell applications. Constraint-based modeling is a useful tool for helping researchers gain insights into the metabolic capabilities of these bacteria. However, Shewanella oneidensis MR-1 is the only strain with a genome-scale metabolic model constructed out of the 22 sequenced Shewanella strains.

  6. Genome-Wide Microarray Expression and Genomic Alterations by Array-CGH Analysis in Neuroblastoma Stem-Like Cells

    PubMed Central

    Martínez-Soto, Soledad; Legarra, Sheila; Pata-Merci, Noémie; Guegan, Justine; Danglot, Giselle; Bernheim, Alain; Meléndez, Bárbara; Rey, Juan A.; Castresana, Javier S.

    2014-01-01

    Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells. PMID:25392930

  7. Identification of an "Exceptional Responder" Cell Line to MEK1 Inhibition: Clinical Implications for MEK-Targeted Therapy | Office of Cancer Genomics

    Cancer.gov

    The identification of somatic genetic alterations that confer sensitivity to pharmacologic inhibitors has led to new cancer therapies. To identify mutations that confer an exceptional dependency, shRNA-based loss-of-function data were analyzed from a dataset of numerous cell lines to reveal genes that are essential in a small subset of cancer cell lines. Once these cell lines were determined, detailed genomic characterization from these cell lines was utilized to ascertain the genomic aberrations that led to this extreme dependency.

  8. Evidence that active demethylation mechanisms maintain the genome of carcinoma in situ cells hypomethylated in the adult testis

    PubMed Central

    Kristensen, D G; Nielsen, J E; Jørgensen, A; Skakkebæk, N E; Rajpert-De Meyts, E; Almstrup, K

    2014-01-01

    Background: Developmental arrest of fetal germ cells may lead to neoplastic transformation and formation of germ cell tumours via carcinoma in situ (CIS) cells. Normal fetal germ cell development requires complete erasure and re-establishment of DNA methylation. In contrast to normal spermatogonia, the genome of CIS cells remains unmethylated in the adult testis. We here investigated the possible active and passive pathways that can sustain the CIS genome hypomethylated in the adult testis. Methods: The levels of 5-methyl-cytosine (5mC) and 5-hydroxy-methyl-cytosine (5hmC) in DNA from micro-dissected CIS cells were assessed by quantitative measurements. The expression of TET1, TET2, APOBEC1, MBD4, APEX1, PARP1, DNMT1, DNMT3A, DNMT3B and DNMT3L in adult testis specimens with CIS and in human fetal testis was investigated by immunohistochemistry and immunofluorescence. Results: DNA from micro-dissected CIS cells contained very low levels of 5hmC produced by ten eleven translocation (TET) enzymes. CIS cells and fetal germ cells expressed the suggested initiator of active demethylation, APOBEC1, and the base excision repair proteins MBD4, APEX1 and PARP1, whereas TETs – the alternative initiators were absent. Both maintenance and de novo methyltransferases were detected in CIS cells. Conclusion: The data are consistent with the presence of an active DNA de-methylation pathway in CIS cells. The hypomethylated genome of CIS cells may contribute to phenotypic plasticity and invasive capabilities of this testicular cancer precursor. PMID:24292451

  9. High, broad, polyfunctional, and durable T cell immune responses induced in mice by a novel hepatitis C virus (HCV) vaccine candidate (MVA-HCV) based on modified vaccinia virus Ankara expressing the nearly full-length HCV genome.

    PubMed

    Gómez, Carmen E; Perdiguero, Beatriz; Cepeda, María Victoria; Mingorance, Lidia; García-Arriaza, Juan; Vandermeeren, Andrea; Sorzano, Carlos Óscar S; Esteban, Mariano

    2013-07-01

    A major goal in the control of hepatitis C infection is the development of a vaccine. Here, we have developed a novel HCV vaccine candidate based on the highly attenuated poxvirus vector MVA (referred to as MVA-HCV) expressing the nearly full-length (7.9-kbp) HCV sequence, with the aim to target almost all of the T and B cell determinants described for HCV. In infected cells, MVA-HCV produces a polyprotein that is subsequently processed into the structural and nonstructural HCV proteins, triggering the cytoplasmic accumulation of dense membrane aggregates. In both C57BL/6 and transgenic HLA-A2-vaccinated mice, MVA-HCV induced high, broad, polyfunctional, and long-lasting HCV-specific T cell immune responses. The vaccine-induced T cell response was mainly mediated by CD8 T cells; however, although lower in magnitude, the CD4(+) T cells were highly polyfunctional. In homologous protocol (MVA-HCV/MVA-HCV) the main CD8(+) T cell target was p7+NS2, whereas in heterologous combination (DNA-HCV/MVA-HCV) the main target was NS3. Antigenic responses were also detected against other HCV proteins (Core, E1-E2, and NS4), but the magnitude of the responses was dependent on the protocol used. The majority of the HCV-induced CD8(+) T cells were triple or quadruple cytokine producers. The MVA-HCV vaccine induced memory CD8(+) T cell responses with an effector memory phenotype. Overall, our data showed that MVA-HCV induced broad, highly polyfunctional, and durable T cell responses of a magnitude and quality that might be associated with protective immunity and open the path for future considerations of MVA-HCV as a prophylactic and/or therapeutic vaccine candidate against HCV.

  10. High, Broad, Polyfunctional, and Durable T Cell Immune Responses Induced in Mice by a Novel Hepatitis C Virus (HCV) Vaccine Candidate (MVA-HCV) Based on Modified Vaccinia Virus Ankara Expressing the Nearly Full-Length HCV Genome

    PubMed Central

    Gómez, Carmen E.; Perdiguero, Beatriz; Cepeda, María Victoria; Mingorance, Lidia; García-Arriaza, Juan; Vandermeeren, Andrea; Sorzano, Carlos Óscar S.

    2013-01-01

    A major goal in the control of hepatitis C infection is the development of a vaccine. Here, we have developed a novel HCV vaccine candidate based on the highly attenuated poxvirus vector MVA (referred to as MVA-HCV) expressing the nearly full-length (7.9-kbp) HCV sequence, with the aim to target almost all of the T and B cell determinants described for HCV. In infected cells, MVA-HCV produces a polyprotein that is subsequently processed into the structural and nonstructural HCV proteins, triggering the cytoplasmic accumulation of dense membrane aggregates. In both C57BL/6 and transgenic HLA-A2-vaccinated mice, MVA-HCV induced high, broad, polyfunctional, and long-lasting HCV-specific T cell immune responses. The vaccine-induced T cell response was mainly mediated by CD8 T cells; however, although lower in magnitude, the CD4+ T cells were highly polyfunctional. In homologous protocol (MVA-HCV/MVA-HCV) the main CD8+ T cell target was p7+NS2, whereas in heterologous combination (DNA-HCV/MVA-HCV) the main target was NS3. Antigenic responses were also detected against other HCV proteins (Core, E1-E2, and NS4), but the magnitude of the responses was dependent on the protocol used. The majority of the HCV-induced CD8+ T cells were triple or quadruple cytokine producers. The MVA-HCV vaccine induced memory CD8+ T cell responses with an effector memory phenotype. Overall, our data showed that MVA-HCV induced broad, highly polyfunctional, and durable T cell responses of a magnitude and quality that might be associated with protective immunity and open the path for future considerations of MVA-HCV as a prophylactic and/or therapeutic vaccine candidate against HCV. PMID:23596307

  11. Whole genome DNA methylation analysis based on high throughput sequencing technology.

    PubMed

    Li, Ning; Ye, Mingzhi; Li, Yingrui; Yan, Zhixiang; Butcher, Lee M; Sun, Jihua; Han, Xu; Chen, Quan; Zhang, Xiuqing; Wang, Jun

    2010-11-01

    There are numerous approaches to decipher a whole genome DNA methylation profile ("methylome"), each varying in cost, throughput and resolution. The gold standard of these methods, whole genome bisulfite-sequencing (BS-seq), involves treatment of DNA with sodium bisulfite combined with subsequent high throughput sequencing. Using BS-seq, we generated a single-base-resolution methylome in human peripheral blood mononuclear cells (in press). This BS-seq map was then used as the reference methylome to compare two alternative sequencing-based methylome assays (performed on the same donor of PBMCs): methylated DNA immunoprecipitation (MeDIP-seq) and methyl-binding protein (MBD-seq). In our analysis, we found that MeDIP-seq and MBD-seq are complementary strategies, with MeDIP-seq more sensitive to highly methylated, high-CpG densities and MDB-seq more sensitive to highly methylated, moderate-CpG densities. Taking into account the size of a mammalian genome and the current expense of sequencing, we feel 3gigabases (Gbp) 45bp paired-end MeDIP-seq or MBD-seq uniquely mapped reads is the minimum requirement and cost-effective strategy for methylome pattern analysis.

  12. Studies on the molecular correlates of genomic stability in rat brain cells following Amalakirasayana therapy.

    PubMed

    Swain, Umakanta; Sindhu, Kiran Kumar; Boda, Ushasri; Pothani, Suresh; Giridharan, Nappan V; Raghunath, Manchala; Rao, Kalluri Subba

    2012-04-01

    Adult Wistar NIN (WNIN) rats (6 months old) of both sexes were orally fed Amalakirasayana at a dose of 4.5 g per kg body weight, five days in a week. The Amalakirasayana was prepared by Arya Vaidya Sala, Kottakkal, Kerala, India, which is considered as gold standard. After 3, 9 and 15 months of such therapeutic regime, rats were sacrificed and various tissues including brain were removed. Isolated cell suspensions of neurons and astroglia were prepared from the cerebral cortex. DNA damage, as a prime indicator of the status of genomic stability was measured in terms of single (SSBs) and double strand breaks (DSBs) through (a). The "comet" assay and (b). The biochemical methods utilizing the unique properties of Escherichia coli DNA polymerase I (pol I) and calf thymus terminal transferase. The results convincingly indicate that while in control animals, there was a distinct increase in DNA damage with age in neurons and astrocytes, rasayana fed animals showed significantly less DNA damage in brain cells demonstrating beneficial effects of Rasayana therapy towards maintenance of genomic stability. DNA-damage may be the proximal cause of aging and strategies to reduce the rate of aging could be based on this fact.

  13. Using FlyBase, a Database of Drosophila Genes & Genomes

    PubMed Central

    Marygold, Steven J.; Crosby, Madeline A.; Goodman, Joshua L.

    2016-01-01

    SUMMARY For nearly 25 years, FlyBase (flybase.org) has provided a freely available online database of biological information about Drosophila species, focusing on the model organism D. melanogaster. The need for a centralized, integrated view of Drosophila research has never been greater as advances in genomic, proteomic and high-throughput technologies add to the quantity and diversity of available data and resources. FlyBase has taken several approaches to respond to these changes in the research landscape. Novel report pages have been generated for new reagent types and physical interaction data; Drosophila models of human disease are now represented and showcased in dedicated Human Disease Model Reports; other integrated reports have been established that bring together related genes, datasets or reagents; Gene Reports have been revised to improve access to new data types and to highlight functional data; links to external sites have been organized and expanded; and new tools have been developed to display and interrogate all these data, including improved batch processing and bulk file availability. In addition, several new community initiatives have served to enhance interactions between researchers and FlyBase, resulting in direct user contributions and improved feedback. This chapter provides an overview of the data content, organization and available tools within FlyBase, focusing on recent improvements. We hope it serves as a guide for our diverse user base, enabling efficient and effective exploration of the database and thereby accelerating research discoveries. PMID:27730573

  14. CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

    PubMed

    Yuen, Kit-San; Chan, Chi-Ping; Wong, Nok-Hei Mickey; Ho, Chau-Ha; Ho, Ting-Hin; Lei, Ting; Deng, Wen; Tsao, Sai Wah; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

    2015-03-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

  15. Limited Genomic Heterogeneity of Circulating Melanoma Cells in Advanced Stage Patients

    PubMed Central

    Ruiz, Carmen; Li, Julia; Luttgen, Madelyn S.; Kolatkar, Anand; Kendall, Jude T.; Flores, Edna; Topp, Zheng; Samlowski, Wolfram E.; McClay, Ed; Bethel, Kelly; Ferrone, Soldano; Hicks, James; Kuhn, Peter

    2015-01-01

    Purpose Circulating melanoma cells (CMCs) constitute a potentially important representation of time-resolved tumor biology in patients. To date, genomic characterization of CMCs has been limited due to the lack of a robust methodology capable of identifying them in a format suitable for downstream characterization. Here, we have developed a methodology to detect intact CMCs that enables phenotypic, morphometric and genomic analysis at the single cell level. Experimental design Blood samples from 40 metastatic melanoma patients and 10 normal blood donors (NBD) were prospectively collected. A panel of 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAb) was used to immunocytochemically label CMCs. Detection was performed by automated digital fluorescence microscopy and multi-parametric computational analysis. Individual CMCs were captured by micromanipulation for whole genome amplification (WGA) and copy number variation (CNV) analysis. Results Based on CSPG4 expression and nuclear size, 1 to 250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5 to 371.5 CMCs/ml). Morphometric analysis revealed that CMCs have a broad spectrum of morphologies and sizes but exhibit a relatively homogeneous nuclear size that was on average 1.5-fold larger than that of surrounding PBMCs. CNV analysis of single CMCs identified deletions of CDKN2A and PTEN, and amplification(s) of TERT, BRAF, KRAS and MDM2. Furthermore, novel chromosomal amplifications in chr12, 17 and 19 were also found. Conclusions Our findings show that CSPG4 expressing CMCs can be found in the majority of advanced melanoma patients. High content analysis of this population may contribute to develop effective therapeutic strategies. PMID:25574741

  16. Integrated genome-based studies of Shewanella ecophysiology

    SciTech Connect

    Segre Daniel; Beg Qasim

    2012-02-14

    This project was a component of the Shewanella Federation and, as such, contributed to the overall goal of applying the genomic tools to better understand eco-physiology and speciation of respiratory-versatile members of Shewanella genus. Our role at Boston University was to perform bioreactor and high throughput gene expression microarrays, and combine dynamic flux balance modeling with experimentally obtained transcriptional and gene expression datasets from different growth conditions. In the first part of project, we designed the S. oneidensis microarray probes for Affymetrix Inc. (based in California), then we identified the pathways of carbon utilization in the metal-reducing marine bacterium Shewanella oneidensis MR-1, using our newly designed high-density oligonucleotide Affymetrix microarray on Shewanella cells grown with various carbon sources. Next, using a combination of experimental and computational approaches, we built algorithm and methods to integrate the transcriptional and metabolic regulatory networks of S. oneidensis. Specifically, we combined mRNA microarray and metabolite measurements with statistical inference and dynamic flux balance analysis (dFBA) to study the transcriptional response of S. oneidensis MR-1 as it passes through exponential, stationary, and transition phases. By measuring time-dependent mRNA expression levels during batch growth of S. oneidensis MR-1 under two radically different nutrient compositions (minimal lactate and nutritionally rich LB medium), we obtain detailed snapshots of the regulatory strategies used by this bacterium to cope with gradually changing nutrient availability. In addition to traditional clustering, which provides a first indication of major regulatory trends and transcription factors activities, we developed and implemented a new computational approach for Dynamic Detection of Transcriptional Triggers (D2T2). This new method allows us to infer a putative topology of transcriptional dependencies

  17. Comparison of whole genome amplification techniques for human single cell exome sequencing

    PubMed Central

    Borgström, Erik; Paterlini, Marta; Mold, Jeff E.; Frisen, Jonas; Lundeberg, Joakim

    2017-01-01

    Background Whole genome amplification (WGA) is currently a prerequisite for single cell whole genome or exome sequencing. Depending on the method used the rate of artifact formation, allelic dropout and sequence coverage over the genome may differ significantly. Results The largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth distribution. These differences also had impact on the downstream variant calling. Conclusively, the products from the AMPLI1 and MALBAC kits were shown to be most similar to the bulk samples and are therefore recommended for WGA of single cells. Discussion In this study four commercial kits for WGA (AMPLI1, MALBAC, Repli-G and PicoPlex) were used to amplify human single cells. The WGA products were exome sequenced together with non-amplified bulk samples from the same source. The resulting data was evaluated in terms of genomic coverage, allelic dropout and SNP calling. PMID:28207771

  18. Genome-based microorganism classification using coalition formulation game.

    PubMed

    Chung, Byung Chang; Han, Gyu-Bum; Cho, Dong-Ho

    2015-01-01

    Genome-based microorganism classification is the one of interesting issues in microorganism taxonomy. However, the advance in sequencing technology requires a low-complex algorithm to process a great amount of bio sequence data. In this paper, we suggest a coalition formation game for microorganism classification, which can be implemented in distributed manner. We extract word frequency feature from microorganism sequences and formulate the coalition game model that considers the distance among word frequency features. Then, we propose a coalition formation algorithm for clustering microorganisms with feature similarity. The performance of proposed algorithm is compared with that of conventional schemes by means of an experiment. According to the result, we showed that the correctness of proposed distributed algorithm is similar to that of conventional centralized schemes.

  19. ArraySearch: A Web-Based Genomic Search Engine.

    PubMed

    Wilson, Tyler J; Ge, Steven X

    2012-01-01

    Recent advances in microarray technologies have resulted in a flood of genomics data. This large body of accumulated data could be used as a knowledge base to help researchers interpret new experimental data. ArraySearch finds statistical correlations between newly observed gene expression profiles and the huge source of well-characterized expression signatures deposited in the public domain. A search query of a list of genes will return experiments on which the genes are significantly up- or downregulated collectively. Searches can also be conducted using gene expression signatures from new experiments. This resource will empower biological researchers with a statistical method to explore expression data from their own research by comparing it with expression signatures from a large public archive.

  20. From genomes to metabolomes: Understanding mechanisms of symbiosis and cell-cell signaling using the archaeal system Ignicoccus-Nanoarchaeum

    SciTech Connect

    Podar, Mircea; Hettich, Robert; Copie, Valerie; Bothner, Brian

    2016-12-16

    (manuscript in preparation) and by characterizing other similar Nanoarchaeota systems. Using a single cell genomics approach we characterized the first terrestrial geothermal Nanoarchaeota system, from Yellowstone National Park. That nanoarchaeon uses a different host, a species of Sulfolobales, and comparative genomics with N. equitans-Ignicoccus allowed us to come up with an evolutionary model for the evolution of this group of organisms across marine and terrestrial ecosystems. Based on metabolic inferences we were also able to isolate in culture the first such terrestrial nanoarchaeal system, also from Yellowstone, which involves a species of Acidilobus. The novel nanoarchaeal system was characterized using proteomics and it helped us better understand the metabolic capabilities of these organisms as well as how co-evolution shapes the genomes of interacting species. It was also one of the very few cases in which prior genomic data was used to successfully design an approach to culture an organism, which remains the gold standard in microbiology research. As a better understanding of interspecies interaction requires multiple model systems, we have pursued identification and genomic characterization or isolation of additional nanoarchaeal systems from geographically and geochemically distinct environments. Two additional nanoarchaeal systems are presently being characterized from hot springs in Yellowstone and Iceland and will be the subject to future publications.

  1. Merkel Cell Polyomavirus Exhibits Dominant Control of the Tumor Genome and Transcriptome in Virus-Associated Merkel Cell Carcinoma

    PubMed Central

    Starrett, Gabriel J.; Marcelus, Christina; Cantalupo, Paul G.; Katz, Joshua P.; Cheng, Jingwei; Akagi, Keiko; Thakuria, Manisha; Rabinowits, Guilherme; Wang, Linda C.; Symer, David E.; Pipas, James M.

    2017-01-01

    ABSTRACT Merkel cell polyomavirus is the primary etiological agent of the aggressive skin cancer Merkel cell carcinoma (MCC). Recent studies have revealed that UV radiation is the primary mechanism for somatic mutagenesis in nonviral forms of MCC. Here, we analyze the whole transcriptomes and genomes of primary MCC tumors. Our study reveals that virus-associated tumors have minimally altered genomes compared to non-virus-associated tumors, which are dominated by UV-mediated mutations. Although virus-associated tumors contain relatively small mutation burdens, they exhibit a distinct mutation signature with observable transcriptionally biased kataegic events. In addition, viral integration sites overlap focal genome amplifications in virus-associated tumors, suggesting a potential mechanism for these events. Collectively, our studies indicate that Merkel cell polyomavirus is capable of hijacking cellular processes and driving tumorigenesis to the same severity as tens of thousands of somatic genome alterations. PMID:28049147

  2. 3D structures of individual mammalian genomes studied by single-cell Hi-C.

    PubMed

    Stevens, Tim J; Lando, David; Basu, Srinjan; Atkinson, Liam P; Cao, Yang; Lee, Steven F; Leeb, Martin; Wohlfahrt, Kai J; Boucher, Wayne; O'Shaughnessy-Kirwan, Aoife; Cramard, Julie; Faure, Andre J; Ralser, Meryem; Blanco, Enrique; Morey, Lluis; Sansó, Miriam; Palayret, Matthieu G S; Lehner, Ben; Di Croce, Luciano; Wutz, Anton; Hendrich, Brian; Klenerman, Dave; Laue, Ernest D

    2017-04-06

    The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.

  3. Somatic cell nuclear transfer: Infinite reproduction of a unique diploid genome

    SciTech Connect

    Kishigami, Satoshi Wakayama, Sayaka; Hosoi, Yoshihiko; Iritani, Akira; Wakayama, Teruhiko

    2008-06-10

    In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the 'Hayflick limit'. However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to 'passage' a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the 'passage' of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.

  4. Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome.

    PubMed

    Kishigami, Satoshi; Wakayama, Sayaka; Hosoi, Yoshihiko; Iritani, Akira; Wakayama, Teruhiko

    2008-06-10

    In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.

  5. Characterizing polymorphic inversions in human genomes by single-cell sequencing.

    PubMed

    Sanders, Ashley D; Hills, Mark; Porubský, David; Guryev, Victor; Falconer, Ester; Lansdorp, Peter M

    2016-11-01

    Identifying genomic features that differ between individuals and cells can help uncover the functional variants that drive phenotypes and disease susceptibilities. For this, single-cell studies are paramount, as it becomes increasingly clear that the contribution of rare but functional cellular subpopulations is important for disease prognosis, management, and progression. Until now, studying these associations has been challenged by our inability to map structural rearrangements accurately and comprehensively. To overcome this, we coupled single-cell sequencing of DNA template strands (Strand-seq) with custom analysis software to rapidly discover, map, and genotype genomic rearrangements at high resolution. This allowed us to explore the distribution and frequency of inversions in a heterogeneous cell population, identify several polymorphic domains in complex regions of the genome, and locate rare alleles in the reference assembly. We then mapped the entire genomic complement of inversions within two unrelated individuals to characterize their distinct inversion profiles and built a nonredundant global reference of structural rearrangements in the human genome. The work described here provides a powerful new framework to study structural variation and genomic heterogeneity in single-cell samples, whether from individuals for population studies or tissue types for biomarker discovery.

  6. Genome profiling of chondrosarcoma using oligonucleotide array-based comparative genomic hybridization.

    PubMed

    Hameed, Meera; Ulger, Celal; Yasar, Duygu; Limaye, Neha; Kurvathi, Rohini; Streck, Deanna; Benevenia, Joseph; Patterson, Francis; Dermody, James J; Toruner, Gokce A

    2009-07-15

    Chondrosarcomas of the bone are malignant hyaline cartilage-forming tumors with an annual incidence rate of 3.6% of all primary bone malignancies in the United States. Specimens of 25 chondrosarcomas (10 grade I, 9 grade II, 1 grade III, and 5 dedifferentiated) from 23 patients were collected from the Department of Pathology at the University Hospital at UMDNJ-New Jersey Medical School from 1996 to 2007. Array-based comparative genomic hybridization (array-CGH) studies were performed on frozen tumor specimens. Recurrent deletions observed in at least in six tumors were 5q13.2, 5q14.2 approximately q21.3, 6q12 approximately q13, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There was a statistically significant association between high-grade tumor (grade III and dedifferentiated) and the recurrent genetic deletions at 5q14.2 approximately q21.3, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There is consistency between increased levels of aneuploidy and the progression of chondrosarcoma from lower to higher grades.

  7. An affinity-based genome walking method to find transgene integration loci in transgenic genome.

    PubMed

    Thirulogachandar, V; Pandey, Prachi; Vaishnavi, C S; Reddy, Malireddy K

    2011-09-15

    Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.

  8. Comparative Single-Cell Genomics of Chloroflexi from the Okinawa Trough Deep-Subsurface Biosphere

    PubMed Central

    Fullerton, Heather

    2016-01-01

    ABSTRACT Chloroflexi small-subunit (SSU) rRNA gene sequences are frequently recovered from subseafloor environments, but the metabolic potential of the phylum is poorly understood. The phylum Chloroflexi is represented by isolates with diverse metabolic strategies, including anoxic phototrophy, fermentation, and reductive dehalogenation; therefore, function cannot be attributed to these organisms based solely on phylogeny. Single-cell genomics can provide metabolic insights into uncultured organisms, like the deep-subsurface Chloroflexi. Nine SSU rRNA gene sequences were identified from single-cell sorts of whole-round core material collected from the Okinawa Trough at Iheya North hydrothermal field as part of Integrated Ocean Drilling Program (IODP) expedition 331 (Deep Hot Biosphere). Previous studies of subsurface Chloroflexi single amplified genomes (SAGs) suggested heterotrophic or lithotrophic metabolisms and provided no evidence for growth by reductive dehalogenation. Our nine Chloroflexi SAGs (seven of which are from the order Anaerolineales) indicate that, in addition to genes for the Wood-Ljungdahl pathway, exogenous carbon sources can be actively transported into cells. At least one subunit for pyruvate ferredoxin oxidoreductase was found in four of the Chloroflexi SAGs. This protein can provide a link between the Wood-Ljungdahl pathway and other carbon anabolic pathways. Finally, one of the seven Anaerolineales SAGs contains a distinct reductive dehalogenase homologous (rdhA) gene. IMPORTANCE Through the use of single amplified genomes (SAGs), we have extended the metabolic potential of an understudied group of subsurface microbes, the Chloroflexi. These microbes are frequently detected in the subsurface biosphere, though their metabolic capabilities have remained elusive. In contrast to previously examined Chloroflexi SAGs, our genomes (several are from the order Anaerolineales) were recovered from a hydrothermally driven system and therefore provide a

  9. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.

    PubMed

    Bankevich, Anton; Nurk, Sergey; Antipov, Dmitry; Gurevich, Alexey A; Dvorkin, Mikhail; Kulikov, Alexander S; Lesin, Valery M; Nikolenko, Sergey I; Pham, Son; Prjibelski, Andrey D; Pyshkin, Alexey V; Sirotkin, Alexander V; Vyahhi, Nikolay; Tesler, Glenn; Alekseyev, Max A; Pevzner, Pavel A

    2012-05-01

    The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

  10. Achilles' heel of pluripotent stem cells: genetic, genomic and epigenetic variations during prolonged culture.

    PubMed

    Rebuzzini, Paola; Zuccotti, Maurizio; Redi, Carlo Alberto; Garagna, Silvia

    2016-07-01

    Pluripotent stem cells differentiate into almost any specialized adult cell type of an organism. PSCs can be derived either from the inner cell mass of a blastocyst-giving rise to embryonic stem cells-or after reprogramming of somatic terminally differentiated cells to obtain ES-like cells, named induced pluripotent stem cells. The potential use of these cells in the clinic, for investigating in vitro early embryonic development or for screening the effects of new drugs or xenobiotics, depends on capability to maintain their genome integrity during prolonged culture and differentiation. Both human and mouse PSCs are prone to genomic and (epi)genetic instability during in vitro culture, a feature that seriously limits their real potential use. Culture-induced variations of specific chromosomes or genes, are almost all unpredictable and, as a whole, differ among independent cell lines. They may arise at different culture passages, suggesting the absence of a safe passage number maintaining genome integrity and rendering the control of genomic stability mandatory since the very early culture passages. The present review highlights the urgency for further studies on the mechanisms involved in determining (epi)genetic and chromosome instability, exploiting the knowledge acquired earlier on other cell types.

  11. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    SciTech Connect

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  12. The population genomics of rhesus macaques (Macaca mulatta) based on whole-genome sequences

    PubMed Central

    Xue, Cheng; Raveendran, Muthuswamy; Harris, R. Alan; Fawcett, Gloria L.; Liu, Xiaoming; White, Simon; Dahdouli, Mahmoud; Rio Deiros, David; Below, Jennifer E.; Salerno, William; Cox, Laura; Fan, Guoping; Ferguson, Betsy; Horvath, Julie; Johnson, Zach; Kanthaswamy, Sree; Kubisch, H. Michael; Liu, Dahai; Platt, Michael; Smith, David G.; Sun, Binghua; Vallender, Eric J.; Wang, Feng; Wiseman, Roger W.; Chen, Rui; Muzny, Donna M.; Gibbs, Richard A.; Yu, Fuli; Rogers, Jeffrey

    2016-01-01

    Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate in biomedical research, have the largest natural geographic distribution of any nonhuman primate, and have been the focus of much evolutionary and behavioral investigation. Consequently, rhesus macaques are one of the most thoroughly studied nonhuman primate species. However, little is known about genome-wide genetic variation in this species. A detailed understanding of extant genomic variation among rhesus macaques has implications for the use of this species as a model for studies of human health and disease, as well as for evolutionary population genomics. Whole-genome sequencing analysis of 133 rhesus macaques revealed more than 43.7 million single-nucleotide variants, including thousands predicted to alter protein sequences, transcript splicing, and transcription factor binding sites. Rhesus macaques exhibit 2.5-fold higher overall nucleotide diversity and slightly elevated putative functional variation compared with humans. This functional variation in macaques provides opportunities for analyses of coding and noncoding variation, and its cellular consequences. Despite modestly higher levels of nonsynonymous variation in the macaques, the estimated distribution of fitness effects and the ratio of nonsynonymous to synonymous variants suggest that purifying selection has had stronger effects in rhesus macaques than in humans. Demographic reconstructions indicate this species has experienced a consistently large but fluctuating population size. Overall, the results presented here provide new insights into the population genomics of nonhuman primates and expand genomic information directly relevant to primate models of human disease. PMID:27934697

  13. The population genomics of rhesus macaques (Macaca mulatta) based on whole-genome sequences.

    PubMed

    Xue, Cheng; Raveendran, Muthuswamy; Harris, R Alan; Fawcett, Gloria L; Liu, Xiaoming; White, Simon; Dahdouli, Mahmoud; Rio Deiros, David; Below, Jennifer E; Salerno, William; Cox, Laura; Fan, Guoping; Ferguson, Betsy; Horvath, Julie; Johnson, Zach; Kanthaswamy, Sree; Kubisch, H Michael; Liu, Dahai; Platt, Michael; Smith, David G; Sun, Binghua; Vallender, Eric J; Wang, Feng; Wiseman, Roger W; Chen, Rui; Muzny, Donna M; Gibbs, Richard A; Yu, Fuli; Rogers, Jeffrey

    2016-12-01

    Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate in biomedical research, have the largest natural geographic distribution of any nonhuman primate, and have been the focus of much evolutionary and behavioral investigation. Consequently, rhesus macaques are one of the most thoroughly studied nonhuman primate species. However, little is known about genome-wide genetic variation in this species. A detailed understanding of extant genomic variation among rhesus macaques has implications for the use of this species as a model for studies of human health and disease, as well as for evolutionary population genomics. Whole-genome sequencing analysis of 133 rhesus macaques revealed more than 43.7 million single-nucleotide variants, including thousands predicted to alter protein sequences, transcript splicing, and transcription factor binding sites. Rhesus macaques exhibit 2.5-fold higher overall nucleotide diversity and slightly elevated putative functional variation compared with humans. This functional variation in macaques provides opportunities for analyses of coding and noncoding variation, and its cellular consequences. Despite modestly higher levels of nonsynonymous variation in the macaques, the estimated distribution of fitness effects and the ratio of nonsynonymous to synonymous variants suggest that purifying selection has had stronger effects in rhesus macaques than in humans. Demographic reconstructions indicate this species has experienced a consistently large but fluctuating population size. Overall, the results presented here provide new insights into the population genomics of nonhuman primates and expand genomic information directly relevant to primate models of human disease.

  14. Improved evidence-based genome-scale metabolic models for maize leaf, embryo, and endosperm

    SciTech Connect

    Seaver, Samuel M.D.; Bradbury, Louis M.T.; Frelin, Océane; Zarecki, Raphy; Ruppin, Eytan; Hanson, Andrew D.; Henry, Christopher S.

    2015-03-10

    There is a growing demand for genome-scale metabolic reconstructions for plants, fueled by the need to understand the metabolic basis of crop yield and by progress in genome and transcriptome sequencing. Methods are also required to enable the interpretation of plant transcriptome data to study how cellular metabolic activity varies under different growth conditions or even within different organs, tissues, and developmental stages. Such methods depend extensively on the accuracy with which genes have been mapped to the biochemical reactions in the plant metabolic pathways. Errors in these mappings lead to metabolic reconstructions with an inflated number of reactions and possible generation of unreliable metabolic phenotype predictions. Here we introduce a new evidence-based genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the gene-reaction associations included within our model. We also present a new approach for applying our model to predict active metabolic genes based on transcriptome data. This method includes a minimal set of reactions associated with low expression genes to enable activity of a maximum number of reactions associated with high expression genes. We apply this method to construct an organ-specific model for the maize leaf, and tissue specific models for maize embryo and endosperm cells. We validate our models using fluxomics data for the endosperm and embryo, demonstrating an improved capacity of our models to fit the available fluxomics data. All models are publicly available via the DOE Systems Biology Knowledgebase and PlantSEED, and our new method is generally applicable for analysis transcript profiles from any plant, paving the way for further in silico studies with a wide variety of plant genomes.

  15. Improved evidence-based genome-scale metabolic models for maize leaf, embryo, and endosperm.

    PubMed

    Seaver, Samuel M D; Bradbury, Louis M T; Frelin, Océane; Zarecki, Raphy; Ruppin, Eytan; Hanson, Andrew D; Henry, Christopher S

    2015-01-01

    There is a growing demand for genome-scale metabolic reconstructions for plants, fueled by the need to understand the metabolic basis of crop yield and by progress in genome and transcriptome sequencing. Methods are also required to enable the interpretation of plant transcriptome data to study how cellular metabolic activity varies under different growth conditions or even within different organs, tissues, and developmental stages. Such methods depend extensively on the accuracy with which genes have been mapped to the biochemical reactions in the plant metabolic pathways. Errors in these mappings lead to metabolic reconstructions with an inflated number of reactions and possible generation of unreliable metabolic phenotype predictions. Here we introduce a new evidence-based genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the gene-reaction associations included within our model. We also present a new approach for applying our model to predict active metabolic genes based on transcriptome data. This method includes a minimal set of reactions associated with low expression genes to enable activity of a maximum number of reactions associated with high expression genes. We apply this method to construct an organ-specific model for the maize leaf, and tissue specific models for maize embryo and endosperm cells. We validate our models using fluxomics data for the endosperm and embryo, demonstrating an improved capacity of our models to fit the available fluxomics data. All models are publicly available via the DOE Systems Biology Knowledgebase and PlantSEED, and our new method is generally applicable for analysis transcript profiles from any plant, paving the way for further in silico studies with a wide variety of plant genomes.

  16. Improved evidence-based genome-scale metabolic models for maize leaf, embryo, and endosperm

    PubMed Central

    Seaver, Samuel M. D.; Bradbury, Louis M. T.; Frelin, Océane; Zarecki, Raphy; Ruppin, Eytan; Hanson, Andrew D.; Henry, Christopher S.

    2015-01-01

    There is a growing demand for genome-scale metabolic reconstructions for plants, fueled by the need to understand the metabolic basis of crop yield and by progress in genome and transcriptome sequencing. Methods are also required to enable the interpretation of plant transcriptome data to study how cellular metabolic activity varies under different growth conditions or even within different organs, tissues, and developmental stages. Such methods depend extensively on the accuracy with which genes have been mapped to the biochemical reactions in the plant metabolic pathways. Errors in these mappings lead to metabolic reconstructions with an inflated number of reactions and possible generation of unreliable metabolic phenotype predictions. Here we introduce a new evidence-based genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the gene-reaction associations included within our model. We also present a new approach for applying our model to predict active metabolic genes based on transcriptome data. This method includes a minimal set of reactions associated with low expression genes to enable activity of a maximum number of reactions associated with high expression genes. We apply this method to construct an organ-specific model for the maize leaf, and tissue specific models for maize embryo and endosperm cells. We validate our models using fluxomics data for the endosperm and embryo, demonstrating an improved capacity of our models to fit the available fluxomics data. All models are publicly available via the DOE Systems Biology Knowledgebase and PlantSEED, and our new method is generally applicable for analysis transcript profiles from any plant, paving the way for further in silico studies with a wide variety of plant genomes. PMID:25806041

  17. Improved evidence-based genome-scale metabolic models for maize leaf, embryo, and endosperm

    DOE PAGES

    Seaver, Samuel M.D.; Bradbury, Louis M.T.; Frelin, Océane; ...

    2015-03-10

    There is a growing demand for genome-scale metabolic reconstructions for plants, fueled by the need to understand the metabolic basis of crop yield and by progress in genome and transcriptome sequencing. Methods are also required to enable the interpretation of plant transcriptome data to study how cellular metabolic activity varies under different growth conditions or even within different organs, tissues, and developmental stages. Such methods depend extensively on the accuracy with which genes have been mapped to the biochemical reactions in the plant metabolic pathways. Errors in these mappings lead to metabolic reconstructions with an inflated number of reactions andmore » possible generation of unreliable metabolic phenotype predictions. Here we introduce a new evidence-based genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the gene-reaction associations included within our model. We also present a new approach for applying our model to predict active metabolic genes based on transcriptome data. This method includes a minimal set of reactions associated with low expression genes to enable activity of a maximum number of reactions associated with high expression genes. We apply this method to construct an organ-specific model for the maize leaf, and tissue specific models for maize embryo and endosperm cells. We validate our models using fluxomics data for the endosperm and embryo, demonstrating an improved capacity of our models to fit the available fluxomics data. All models are publicly available via the DOE Systems Biology Knowledgebase and PlantSEED, and our new method is generally applicable for analysis transcript profiles from any plant, paving the way for further in silico studies with a wide variety of plant genomes.« less

  18. Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

    PubMed Central

    Guo, Michael H.; Nandakumar, Satish K.; Ulirsch, Jacob C.; Zekavat, Seyedeh M.; Buenrostro, Jason D.; Natarajan, Pradeep; Salem, Rany M.; Chiarle, Roberto; Mitt, Mario; Kals, Mart; Pärn, Kalle; Fischer, Krista; Milani, Lili; Mägi, Reedik; Palta, Priit; Gabriel, Stacey B.; Metspalu, Andres; Lander, Eric S.; Kathiresan, Sekar; Hirschhorn, Joel N.; Esko, Tõnu; Sankaran, Vijay G.

    2017-01-01

    Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance. PMID:28031487

  19. Repetitive element signature-based visualization, distance computation, and classification of 1766 microbial genomes.

    PubMed

    Lee, Kang-Hoon; Shin, Kyung-Seop; Lim, Debora; Kim, Woo-Chan; Chung, Byung Chang; Han, Gyu-Bum; Roh, Jeongkyu; Cho, Dong-Ho; Cho, Kiho

    2015-07-01

    The genomes of living organisms are populated with pleomorphic repetitive elements (REs) of varying densities. Our hypothesis that genomic RE landscapes are species/strain/individual-specific was implemented into the Genome Signature Imaging system to visualize and compute the RE-based signatures of any genome. Following the occurrence profiling of 5-nucleotide REs/words, the information from top-50 frequency words was transformed into a genome-specific signature and visualized as Genome Signature Images (GSIs), using a CMYK scheme. An algorithm for computing distances among GSIs was formulated using the GSIs' variables (word identity, frequency, and frequency order). The utility of the GSI-distance computation system was demonstrated with control genomes. GSI-based computation of genome-relatedness among 1766 microbes (117 archaea and 1649 bacteria) identified their clustering patterns; although the majority paralleled the established classification, some did not. The Genome Signature Imaging system, with its visualization and distance computation functions, enables genome-scale evolutionary studies involving numerous genomes with varying sizes.

  20. Genome-based approaches to the authentication of medicinal plants.

    PubMed

    Sucher, Nikolaus J; Carles, Maria C

    2008-05-01

    Medicinal plants are the source of a large number of essential drugs in Western medicine and are the basis of herbal medicine, which is not only the primary source of health care for most of the world's population living in developing countries but also enjoys growing popularity in developed countries. The increased demand for botanical products is met by an expanding industry and accompanied by calls for assurance of quality, efficacy and safety. Plants used as drugs, dietary supplements and herbal medicines are identified at the species level. Unequivocal identification is a critical step at the beginning of an extensive process of quality assurance and is of importance for the characterization of the genetic diversity, phylogeny and phylogeography as well as the protection of endangered species. DNA-based methods have been developed for the identification of medicinal plants. Nuclear and chloroplast DNA is amplified by the polymerase chain reaction and the reaction products are analyzed by gel electrophoresis, sequencing, or hybridization with species-specific probes. Genomic fingerprinting can differentiate between individuals, species and populations and is useful for the detection of the homogeneity of the samples and presence of adulterants. Although sequences from single chloroplast or nuclear genes have been useful for differentiation of species, phylogenetic studies often require consideration of DNA sequence data from more than one gene or genomic region. Phytochemical and genetic data are correlated but only the latter normally allow for differentiation at the species level. The generation of molecular "barcodes" of medicinal plants will be worth the concerted effort of the medicinal plant research community and contribute to the ongoing effort of defining barcodes for every species on earth.

  1. Cell-Type-Specific Genome-wide Expression Profiling after Laser Capture Microdissection of Living Tissue

    SciTech Connect

    Marchetti, F; Manohar, C F

    2005-02-09

    The purpose of this technical feasibility study was to develop and evaluate robust microgenomic tools for investigations of genome-wide expression of very small numbers of cells isolated from whole tissue sections. Tissues contain large numbers of cell-types that play varied roles in organ function and responses to endogenous and exogenous toxicants whether bacterial, viral, chemical or radiation. Expression studies of whole tissue biopsy are severely limited because heterogeneous cell-types result in an averaging of molecular signals masking subtle but important changes in gene expression in any one cell type(s) or group of cells. Accurate gene expression analysis requires the study of specific cell types in their tissue environment but without contamination from surrounding cells. Laser capture microdissection (LCM) is a new technology to isolate morphologically distinct cells from tissue sections. Alternative methods are available for isolating single cells but not yet for their reliable genome-wide expression analyses. The tasks of this feasibility project were to: (1) Develop efficient protocols for laser capture microdissection of cells from tissues identified by antibody label, or morphological stain. (2) Develop reproducible gene-transcript analyses techniques for single cell-types and determine the numbers of cells needed for reliable genome-wide analyses. (3) Validate the technology for epithelial and endothelial cells isolated from the gastrointestinal tract of mice.

  2. GeNemo: a search engine for web-based functional genomic data.

    PubMed

    Zhang, Yongqing; Cao, Xiaoyi; Zhong, Sheng

    2016-07-08

    A set of new data types emerged from functional genomic assays, including ChIP-seq, DNase-seq, FAIRE-seq and others. The results are typically stored as genome-wide intensities (WIG/bigWig files) or functional genomic regions (peak/BED files). These data types present new challenges to big data science. Here, we present GeNemo, a web-based search engine for functional genomic data. GeNemo searches user-input data against online functional genomic datasets, including the entire collection of ENCODE and mouse ENCODE datasets. Unlike text-based search engines, GeNemo's searches are based on pattern matching of functional genomic regions. This distinguishes GeNemo from text or DNA sequence searches. The user can input any complete or partial functional genomic dataset, for example, a binding intensity file (bigWig) or a peak file. GeNemo reports any genomic regions, ranging from hundred bases to hundred thousand bases, from any of the online ENCODE datasets that share similar functional (binding, modification, accessibility) patterns. This is enabled by a Markov Chain Monte Carlo-based maximization process, executed on up to 24 parallel computing threads. By clicking on a search result, the user can visually compare her/his data with the found datasets and navigate the identified genomic regions. GeNemo is available at www.genemo.org.

  3. Application of single-cell genomics in cancer: promise and challenges.

    PubMed

    Wills, Quin F; Mead, Adam J

    2015-10-15

    Recent advances in single-cell genomics are opening up unprecedented opportunities to transform cancer genomics. While bulk tissue genomic analysis across large populations of tumour cells has provided key insights into cancer biology, this approach does not provide the resolution that is critical for understanding the interaction between different genetic events within the cellular hierarchy of the tumour during disease initiation, evolution, relapse and metastasis. Single-cell genomic approaches are uniquely placed to definitively unravel complex clonal structures and tissue hierarchies, account for spatiotemporal cell interactions and discover rare cells that drive metastatic disease, drug resistance and disease progression. Here we present five challenges that need to be met for single-cell genomics to fulfil its potential as a routine tool alongside bulk sequencing. These might be thought of as being challenges related to samples (processing and scale for analysis), sensitivity and specificity of mutation detection, sources of heterogeneity (biological and technical), synergies (from data integration) and systems modelling. We discuss these in the context of recent advances in technologies and data modelling, concluding with implications for moving cancer research into the clinic.

  4. Scalable whole-genome single-cell library preparation without preamplification.

    PubMed

    Zahn, Hans; Steif, Adi; Laks, Emma; Eirew, Peter; VanInsberghe, Michael; Shah, Sohrab P; Aparicio, Samuel; Hansen, Carl L

    2017-02-01

    Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated 'bulk-equivalent' genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.

  5. [Cell based therapy for COPD].

    PubMed

    Kubo, Hiroshi

    2007-04-01

    To develop a new cell based therapy for chronic obstructive pulmonary disease (COPD), we need to understand 1) the role of tissue-specific and bone marrow-derived stem cells, 2) extracellular matrix, and 3) growth factors. Recently, bronchioalveolar stem cells were identified in murine distal lungs. Impairment of these stem cells may cause improper lung repair after inflammation, resulting in pulmonary emphysema. Bone marrow-derived cells are necessary to repair injured lungs. However, the long term role of these cells is not understood yet. Although we need more careful analysis and additional experiments, growth factors, such as hepatocyte growth factor, are good candidates for the new cell based therapy for COPD. Lung was believed as a non-regenerative organ. Based on these recent reports about lung regeneration and stem cells, however, new strategies to treat COPD and a new point of view to understand the pathophysiology of COPD are rising.

  6. The FlyBase database of the Drosophila genome projects andcommunity literature

    SciTech Connect

    Gelbart, William; Bayraktaroglu, Leyla; Bettencourt, Brian; Campbell, Kathy; Crosby, Madeline; Emmert, David; Hradecky, Pavel; Huang,Yanmei; Letovsky, Stan; Matthews, Beverly; Russo, Susan; Schroeder,Andrew; Smutniak, Frank; Zhou, Pinglei; Zytkovicz, Mark; Ashburner,Michael; Drysdale, Rachel; de Grey, Aubrey; Foulger, Rebecca; Millburn,Gillian; Yamada, Chihiro; Kaufman, Thomas; Matthews, Kathy; Gilbert, Don; Grumbling, Gary; Strelets, Victor; Shemen, C.; Rubin, Gerald; Berman,Brian; Frise, Erwin; Gibson, Mark; Harris, Nomi; Kaminker, Josh; Lewis,Suzanna; Marshall, Brad; Misra, Sima; Mungall, Christopher; Prochnik,Simon; Richter, John; Smith, Christopher; Shu, ShengQiang; Tupy,Jonathan; Wiel, Colin

    2002-09-16

    FlyBase (http://flybase.bio.indiana.edu/) provides an integrated view of the fundamental genomic and genetic data on the major genetic model Drosophila melanogaster and related species. FlyBase has primary responsibility for the continual reannotation of the D.melanogaster genome. The ultimate goal of the reannotation effort is to decorate the euchromatic sequence of the genome with as much biological information as is available from the community and from the major genome project centers. A complete revision of the annotations of the now-finished euchromatic genomic sequence has been completed. There are many points of entry to the genome within FlyBase, most notably through maps, gene products and ontologies, structured phenotypic and gene expression data, and anatomy.

  7. Systematic Prioritization of Druggable Mutations in ∼5000 Genomes Across 16 Cancer Types Using a Structural Genomics-based Approach*

    PubMed Central

    Zhao, Junfei; Cheng, Feixiong; Wang, Yuanyuan; Arteaga, Carlos L.; Zhao, Zhongming

    2016-01-01

    A massive amount of somatic mutations has been cataloged in large-scale projects such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium projects. The majority of the somatic mutations found in tumor genomes are neutral 'passenger' rather than damaging “driver” mutations. Now, understanding their biological consequences and prioritizing them for druggable targets are urgently needed. Thanks to the rapid advances in structural genomics technologies (e.g. X-ray), large-scale protein structural data has now been made available, providing critical information for deciphering functional roles of mutations in cancer and prioritizing those alterations that may mediate drug binding at the atom resolution and, as such, be druggable targets. We hypothesized that mutations at protein–ligand binding-site residues are likely to be druggable targets. Thus, to prioritize druggable mutations, we developed SGDriver, a structural genomics-based method incorporating the somatic missense mutations into protein–ligand binding-site residues using a Bayes inference statistical framework. We applied SGDriver to 746,631 missense mutations observed in 4997 tumor-normal pairs across 16 cancer types from The Cancer Genome Atlas. SGDriver detected 14,471 potential druggable mutations in 2091 proteins (including 1,516 recurrently mutated proteins) across 3558 cancer genomes (71.2%), and further identified 298 proteins harboring mutations that were significantly enriched at protein–ligand binding-site residues (adjusted p value < 0.05). The identified proteins are significantly enriched in both oncoproteins and tumor suppressors. The follow-up drug-target network analysis suggested 98 known and 126 repurposed druggable anticancer targets (e.g. SPOP and NR3C1). Furthermore, our integrative analysis indicated that 13% of patients might benefit from current targeted therapy, and this –proportion would increase to 31% when considering drug repositioning

  8. Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consortium.

    PubMed

    Salomonis, Nathan; Dexheimer, Phillip J; Omberg, Larsson; Schroll, Robin; Bush, Stacy; Huo, Jeffrey; Schriml, Lynn; Ho Sui, Shannan; Keddache, Mehdi; Mayhew, Christopher; Shanmukhappa, Shiva Kumar; Wells, James; Daily, Kenneth; Hubler, Shane; Wang, Yuliang; Zambidis, Elias; Margolin, Adam; Hide, Winston; Hatzopoulos, Antonis K; Malik, Punam; Cancelas, Jose A; Aronow, Bruce J; Lutzko, Carolyn

    2016-07-12

    The rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from these analyses, including powerful tools, are interactively accessible from a new online portal at https://www.synapse.org to serve as a reusable resource for the stem cell community.

  9. Cell-Free DNA Provides a Good Representation of the Tumor Genome Despite Its Biased Fragmentation Patterns

    PubMed Central

    Zhu, Liangjun; Wu, Xue; Bao, Hua; Wang, Xiaonan; Chang, Zhili; Wang, Zhenxin

    2017-01-01

    Cell-free DNA (cfDNA) is short, extracellular, fragmented double-stranded DNA found in plasma. Plasma of patients with solid tumor has been found to show significantly increased quantities of cfDNA. Although currently poorly understood, the mechanism of cfDNA generation is speculated to be a product of genomic DNA fragmentation during cellular apoptosis and necrosis. Sequencing of cfDNA with tumor origin has identified tumor biomarkers, elucidating molecular pathology and assisting in accurate diagnosis. In this study, we performed whole-genome sequencing ofcfDNA samples with matching tumor and whole blood samples from five patients diagnosed with stage IV gastric or lung cancer. We analyzed the coverage spectrum of the human genome in our cfDNA samples. cfDNA exhibited no large regions with significant under-coverage, although we observed unbalanced coverage depth in cfDNA at transcription start sites and exon boundaries as a consequence of biased fragmentation due to ordered nucleosome positioning. We also analyzed the copy number variant status based on the whole-genome sequencing results and found high similarity between copy number profile constructed from tumor samples and cfDNA samples. Overall, we conclude that cfDNA comprises a good representation of the tumor genome in late stage gastric and lung cancer. PMID:28046008

  10. A whole-genome analysis of a transgenic rice seed-based edible vaccine against cedar pollen allergy.

    PubMed

    Kawakatsu, Taiji; Kawahara, Yoshihiro; Itoh, Takeshi; Takaiwa, Fumio

    2013-12-01

    Genetic modification (GM) by Agrobacterium-mediated transformation is a robust and widely employed method to confer new traits to crops. In this process, a transfer DNA is delivered into the host genome, but it is still unclear how the host genome is altered by this event at single-base resolution. To decipher genomic discrepancy between GM crops and their host, we conducted whole-genome sequencing of a transgenic rice line OSCR11. This rice line expresses a seed-based edible vaccine containing two major pollen allergens, Cry j 1 and Cry j 2, against Japanese cedar pollinosis. We revealed that genetic differences between OSCR11 and its host a123 were significantly less than those between a123 and its precedent cultivar Koshihikari. The pattern of nucleotide base substitution in OSCR11, relative to a123, was consistent with somaclonal variation. Mutations in OSCR11 probably occurred during the cell culture steps. In addition, strand-specific mRNA-Seq revealed similar transcriptomes of a123 and OSCR11, supporting genomic integrity between them.

  11. Single-cell sequencing of Thiomargarita reveals genomic flexibility for adaptation to dynamic redox conditions

    DOE PAGES

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; ...

    2016-06-21

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiornargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of amore » chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na+-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In conclusion, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur

  12. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions

    PubMed Central

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N.; Flood, Beverly E.; Bailey, Jake V.; Mußmann, Marc

    2016-01-01

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming “Candidatus Thiomargarita nelsonii Thio36”, and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. “Ca. T. nelsonii Thio36” is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that “Ca. T. nelsonii Thio36” can function as a chemolithoautotroph. Carbon can be fixed via the Calvin–Benson–Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, “Ca. T. nelsonii Thio36” also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na+-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae “Ca. T. nelsonii Thio36” encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of “Ca. T. nelsonii Thio36” provides additional insight into the ecology of

  13. Accessing the Genomic Effects of Naked Nanoceria in Murine Neuronal Cells

    PubMed Central

    Lee, Tin-Lap; Raitano, Joan M.; Rennert, Owen M; Chan, Siu-Wai; Chan, Wai-Yee

    2011-01-01

    Cerium oxide nanoparticles (nanoceria) are versatile engineered nanoparticles (ENPs) due to their unique redox properties. We and others have previously demonstrated naked nanoceria could act as antioxidants to protect cells against oxidative damage. While the redox properties may be beneficial, the genome-wide effects of nanoceria on gene transcription and associated biological processes remain elusive. Here we applied functional genomic approach to examine the genome-wide effects of nanoceria on global gene transcription and cellular functions in mouse neuronal cells. Importantly, we demonstrated nanoceria induced chemical- and size-specific changes in the murine neuronal cell transcriptome. The nanoceria specially contributed more than 83% of uniquely altered gene population and associate with a unique spectrum of genes related to neurological disease, cell cycle control and growth. These observations suggest an in-depth assessment of potential health effects of naked nanoceria and other naked nanoparticles is both necessary and imminent. PMID:21889474

  14. Regulation and Function of Cdt1; A Key Factor in Cell Proliferation and Genome Stability

    PubMed Central

    Pozo, Pedro N.; Cook, Jeanette Gowen

    2016-01-01

    Successful cell proliferation requires efficient and precise genome duplication followed by accurate chromosome segregation. The Cdc10-dependent transcript 1 protein (Cdt1) is required for the first step in DNA replication, and in human cells Cdt1 is also required during mitosis. Tight cell cycle controls over Cdt1 abundance and activity are critical to normal development and genome stability. We review here recent advances in elucidating Cdt1 molecular functions in both origin licensing and kinetochore–microtubule attachment, and we describe the current understanding of human Cdt1 regulation. PMID:28025526

  15. Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs).

    PubMed

    Taheri-Ghahfarokhi, Amir; Malaver-Ortega, Luis F; Sumer, Huseyin

    2015-01-01

    Interest is increasing in transcription activator-like effector nucleases (TALENs) as a tool to introduce targeted double-strand breaks into the large genomes of human and animal cell lines. The produced DNA lesions stimulate DNA repair pathways, error-prone but dominant non-homologous end joining (NHEJ) and accurate but less occurring homology-directed repair (HDR), and as a result targeted genes can be modified. Here, we describe a modified Golden-Gate cloning method for generating TALENs and also details for targeting genes in mouse embryonic stem cells. The protocol described here can be used for modifying the genome of a broad range of pluripotent cell lines.

  16. Accessing integrated genomic data using GenoBase: A tutorial, Part 1

    SciTech Connect

    Overbeek, R.; Price, M.

    1993-01-01

    GenoBase integrates genomic information from many existing databases, offering convenient access to the curated data. This document is the first part of a two-part tutorial on how to use GenoBase for accessing integrated genomic data.

  17. Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation.

    PubMed

    Stumpf, F; Schoendube, J; Gross, A; Rath, C; Niekrawietz, S; Koltay, P; Roth, G

    2015-07-15

    Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cell line) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10µm diameter) resulting in a single bead delivery in 27 out of 28 cases and relative positional precision of ±350µm at a printing distance of 6mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3±2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing.

  18. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

    NASA Technical Reports Server (NTRS)

    Romanov, S. R.; Kozakiewicz, B. K.; Holst, C. R.; Stampfer, M. R.; Haupt, L. M.; Tlsty, T. D.

    2001-01-01

    Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

  19. Genome-derived cytosolic DNA contributes to type I interferon expression and immunogenicity of B-cell lymphoma cells.

    PubMed

    Shen, Yu J; Ho, Samantha S W; Tan, Nikki Y; Koo, Christine Xing'Er; Khatoo, Muznah; Cheung, Florence S G; Gasser, Stephan

    2015-12-01

    We recently provided evidence that genome-derived DNA is present in the cytosol of many tumor cells. Genomic loci that give rise to cytosolic DNA can potentially form non-B DNA structures including triple-stranded RNA:DNA structures (R-loops). The RNA:DNA-specific endonuclease RNaseh1 reduced the levels of cytosolic DNA and type I interferon-dependent rejection of B-cell lymphoma suggesting that cytosolic DNA may contribute to immune surveillance of B-cell lymphoma.

  20. The genome landscape of the african green monkey kidney-derived vero cell line.

    PubMed

    Osada, Naoki; Kohara, Arihiro; Yamaji, Toshiyuki; Hirayama, Noriko; Kasai, Fumio; Sekizuka, Tsuyoshi; Kuroda, Makoto; Hanada, Kentaro

    2014-12-01

    Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes were identified in the 2.97-Gb genome sequence. A homozygous ∼9-Mb deletion on chromosome 12 caused the loss of the type I interferon gene cluster and cyclin-dependent kinase inhibitor genes in Vero cells. In addition, an ∼59-Mb loss of heterozygosity around this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. Moreover, a genomic analysis of Vero cells revealed a female Chlorocebus sabaeus origin and proviral variations of the endogenous simian type D retrovirus. These results revealed the genomic basis for the non-tumourigenic permanent Vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines and developing new tools in the quality control of Vero cells.

  1. Development of Fibroblast Cell Lines From the Cow Used to Sequence the Bovine Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two cell lines, designated MARC.BGCF.2 and MARC.BGCF.1-3, were initiated from skin biopsies obtained from the Hereford cow whose DNA was used in sequencing the bovine genome. These cell lines were submitted to American Type Culture Collection (ATCC, Manassas, VA, USA) and will be made publicly avai...

  2. Editing genomic DNA in cancer cells with high genetic variance: benefit or risk?

    PubMed

    Wang, Lin; Wang, Yixiang; Guo, Chuanbin

    2014-05-01

    The generation of stably-transfected cell lines is a common and very important technology in cancer science. Considerable knowledge in the field of life sciences has been gained through the modification of the genetic code. However, there is a risk in evaluating exogenous gene function through editing genomic DNA in a cancer cell with high genetic variance. In the present study, we showed that genomic DNA status should be considered when evaluating the exogenous gene function in a cancer cell line with high variant genome through stable transfection technology, immunostaining, wound healing assay, transwell invasion assay, real-time PCR, western blot and karyotyping analysis. Our results showed that the S100P expression level was not related to the migration and invasion abilities in these stably transfected cell lines derived from a human salivary adenoid cystic carcinoma cell line SACC-83. The MMP expression pattern was detected by western blot analysis which matched the biological behaviors in these cells. The genomic analysis showed that SACC-83 presented hypotetraploid karyotyping with high variance. Our data indicated that establishment of stable transgenic cancer cell lines should consider the status of genetic variance in a cancer cell to avoid any biased conclusion.

  3. [CRISPR/Cas9-based genome editing systems and the analysis of targeted genome mutations in plants].

    PubMed

    Xingliang, Ma; Yaoguang, Liu

    2016-02-01

    Targeted genomic editing technologies use programmable DNA nucleases to cleave genomic target sites, thus inducing targeted mutations in the genomes. The newly prevailed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system that consists of the Cas9 nuclease and single guide RNA (sgRNA) has the advantages of simplicity and high efficiency as compared to other programmable DNA nuclease systems such as zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). Currently, a number of cases have been reported on the application of the CRISPR/Cas9 genomic editing technology in plants. In this review, we summarize the strategies for preparing the Cas9 and sgRNA expression constructs, the transformation method for obtaining targeted mutations, the efficiency and features of the resulting mutations and the methods for detecting or genotyping of the mutation sites. We also discuss the existing problems and perspectives of CRISPR/Cas9-based genomic editing in plants.

  4. Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

    PubMed

    Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

    2003-07-15

    Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

  5. Recent advances in genome-based polyketide discovery.

    PubMed

    Helfrich, Eric J N; Reiter, Silke; Piel, Jörn

    2014-10-01

    Polyketides are extraordinarily diverse secondary metabolites of great pharmacological value and with interesting ecological functions. The post-genomics era has led to fundamental changes in natural product research by inverting the workflow of secondary metabolite discovery. As opposed to traditional bioactivity-guided screenings, genome mining is an in silico method to screen and analyze sequenced genomes for natural product biosynthetic gene clusters. Since genes for known compounds can be recognized at the early computational stage, genome mining presents an opportunity for dereplication. This review highlights recent progress in bioinformatics, pathway engineering and chemical analytics to extract the biosynthetic secrets hidden in the genome of both well-known natural product sources as well as previously neglected bacteria.

  6. Research progress of plant population genomics based on high-throughput sequencing.

    PubMed

    Yunsheng, Wang

    2016-08-01

    Population genomics, a new paradigm for population genetics, combine the concepts and techniques of genomics with the theoretical system of population genetics and improve our understanding of microevolution through identification of site-specific effect and genome-wide effects using genome-wide polymorphic sites genotypeing. With the appearance and improvement of the next generation high-throughput sequencing technology, the numbers of plant species with complete genome sequences increased rapidly and large scale resequencing has also been carried out in recent years. Parallel sequencing has also been done in some plant species without complete genome sequences. These studies have greatly promoted the development of population genomics and deepened our understanding of the genetic diversity, level of linking disequilibium, selection effect, demographical history and molecular mechanism of complex traits of relevant plant population at a genomic level. In this review, I briely introduced the concept and research methods of population genomics and summarized the research progress of plant population genomics based on high-throughput sequencing. I also discussed the prospect as well as existing problems of plant population genomics in order to provide references for related studies.

  7. Mitochondrial Transcription Factor A and Mitochondrial Genome as Molecular Targets for Cisplatin-Based Cancer Chemotherapy.

    PubMed

    Kohno, Kimitoshi; Wang, Ke-Yong; Takahashi, Mayu; Kurita, Tomoko; Yoshida, Yoichiro; Hirakawa, Masakazu; Harada, Yoshikazu; Kuma, Akihiro; Izumi, Hiroto; Matsumoto, Shinji

    2015-08-20

    Mitochondria are important cellular organelles that function as control centers of the energy supply for highly proliferative cancer cells and regulate apoptosis after cancer chemotherapy. Cisplatin is one of the most important chemotherapeutic agents and a key drug in therapeutic regimens for a broad range of solid tumors. Cisplatin may directly interact with mitochondria, which can induce apoptosis. The direct interactions between cisplatin and mitochondria may account for our understanding of the clinical activity of cisplatin and development of resistance. However, the basis for the roles of mitochondria under treatment with chemotherapy is poorly understood. In this review, we present novel aspects regarding the unique characteristics of the mitochondrial genome in relation to the use of platinum-based chemotherapy and describe our recent work demonstrating the importance of the mitochondrial transcription factor A (mtTFA) expression in cancer cells.

  8. Role of microRNAs and DNA Methyltransferases in Transmitting Induced Genomic Instability between Cell Generations

    PubMed Central

    Huumonen, Katriina; Korkalainen, Merja; Viluksela, Matti; Lahtinen, Tapani; Naarala, Jonne; Juutilainen, Jukka

    2014-01-01

    There is limited understanding of how radiation or chemicals induce genomic instability, and how the instability is epigenetically transmitted to the progeny of exposed cells or organisms. Here, we measured the expression of microRNAs (miRNAs) and DNA methyltransferases (DNMTs) in murine embryonal fibroblasts exposed to ionizing radiation or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which were previously shown to induce genomic instability in this cell line. Cadmium was used as a reference agent that does not induce genomic instability in our experimental model. Measurements at 8 and 15 days after exposure did not identify any such persistent changes that could be considered as signals transmitting genomic instability to the progeny of exposed cells. However, measurements at 2 days after exposure revealed findings that may reflect initial stages of genomic instability. Changes that were common to TCDD and two doses of radiation (but not to cadmium) included five candidate signature miRNAs and general up-regulation of miRNA expression. Expression of DNMT3a, DNMT3b, and DNMT2 was suppressed by cadmium but not by TCDD or radiation, consistently with the hypothesis that sufficient expression of DNMTs is necessary in the initial phase of induced genomic instability. PMID:25309892

  9. The Transient Inactivation of the Master Cell Cycle Phosphatase Cdc14 Causes Genomic Instability in Diploid Cells of Saccharomyces cerevisiae

    PubMed Central

    Quevedo, Oliver; Ramos-Pérez, Cristina; Petes, Thomas D.; Machín, Félix

    2015-01-01

    Genomic instability is a common feature found in cancer cells . Accordingly, many tumor suppressor genes identified in familiar cancer syndromes are involved in the maintenance of the stability of the genome during every cell division and are commonly referred to as caretakers. Inactivating mutations and epigenetic silencing of caretakers are thought to be the most important mechanisms that explain cancer-related genome instability. However, little is known of whether transient inactivation of caretaker proteins could trigger genome instability and, if so, what types of instability would occur. In this work, we show that a brief and reversible inactivation, during just one cell cycle, of the key phosphatase Cdc14 in the model organism Saccharomyces cerevisiae is enough to result in diploid cells with multiple gross chromosomal rearrangements and changes in ploidy. Interestingly, we observed that such transient loss yields a characteristic fingerprint whereby trisomies are often found in small-sized chromosomes, and gross chromosome rearrangements, often associated with concomitant loss of heterozygosity, are detected mainly on the ribosomal DNA-bearing chromosome XII. Taking into account the key role of Cdc14 in preventing anaphase bridges, resetting replication origins, and controlling spindle dynamics in a well-defined window within anaphase, we speculate that the transient loss of Cdc14 activity causes cells to go through a single mitotic catastrophe with irreversible consequences for the genome stability of the progeny. PMID:25971663

  10. The Transient Inactivation of the Master Cell Cycle Phosphatase Cdc14 Causes Genomic Instability in Diploid Cells of Saccharomyces cerevisiae.

    PubMed

    Quevedo, Oliver; Ramos-Pérez, Cristina; Petes, Thomas D; Machín, Félix

    2015-07-01

    Genomic instability is a common feature found in cancer cells . Accordingly, many tumor suppressor genes identified in familiar cancer syndromes are involved in the maintenance of the stability of the genome during every cell division and are commonly referred to as caretakers. Inactivating mutations and epigenetic silencing of caretakers are thought to be the most important mechanisms that explain cancer-related genome instability. However, little is known of whether transient inactivation of caretaker proteins could trigger genome instability and, if so, what types of instability would occur. In this work, we show that a brief and reversible inactivation, during just one cell cycle, of the key phosphatase Cdc14 in the model organism Saccharomyces cerevisiae is enough to result in diploid cells with multiple gross chromosomal rearrangements and changes in ploidy. Interestingly, we observed that such transient loss yields a characteristic fingerprint whereby trisomies are often found in small-sized chromosomes, and gross chromosome rearrangements, often associated with concomitant loss of heterozygosity, are detected mainly on the ribosomal DNA-bearing chromosome XII. Taking into account the key role of Cdc14 in preventing anaphase bridges, resetting replication origins, and controlling spindle dynamics in a well-defined window within anaphase, we speculate that the transient loss of Cdc14 activity causes cells to go through a single mitotic catastrophe with irreversible consequences for the genome stability of the progeny.

  11. Phenotype prediction based on genome-wide DNA methylation data

    PubMed Central

    2014-01-01

    Background DNA methylation (DNAm) has important regulatory roles in many biological processes and diseases. It is the only epigenetic mark with a clear mechanism of mitotic inheritance and the only one easily available on a genome scale. Aberrant cytosine-phosphate-guanine (CpG) methylation has been discussed in the context of disease aetiology, especially cancer. CpG hypermethylation of promoter regions is often associated with silencing of tumour suppressor genes and hypomethylation with activation of oncogenes. Supervised principal component analysis (SPCA) is a popular machine learning method. However, in a recent application to phenotype prediction from DNAm data SPCA was inferior to the specific method EVORA. Results We present Model-Selection-SPCA (MS-SPCA), an enhanced version of SPCA. MS-SPCA applies several models that perform well in the training data to the test data and selects the very best models for final prediction based on parameters of the test data. We have applied MS-SPCA for phenotype prediction from genome-wide DNAm data. CpGs used for prediction are selected based on the quantification of three features of their methylation (average methylation difference, methylation variation difference and methylation-age-correlation). We analysed four independent case–control datasets that correspond to different stages of cervical cancer: (i) cases currently cytologically normal, but will later develop neoplastic transformations, (ii, iii) cases showing neoplastic transformations and (iv) cases with confirmed cancer. The first dataset was split into several smaller case–control datasets (samples either Human Papilloma Virus (HPV) positive or negative). We demonstrate that cytology normal HPV+ and HPV- samples contain DNAm patterns which are associated with later neoplastic transformations. We present evidence that DNAm patterns exist in cytology normal HPV- samples that (i) predispose to neoplastic transformations after HPV infection and (ii

  12. Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains.

    PubMed

    Van der Aa, Niels; Cheng, Jiqiu; Mateiu, Ligia; Zamani Esteki, Masoud; Kumar, Parveen; Dimitriadou, Eftychia; Vanneste, Evelyne; Moreau, Yves; Vermeesch, Joris Robert; Voet, Thierry

    2013-04-01

    Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell's copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.

  13. Integration of the full-length HPV16 genome in cervical cancer and Caski and Siha cell lines and the possible ways of HPV integration.

    PubMed

    Xu, Feng; Cao, Meng; Shi, Qinfeng; Chen, Hongwei; Wang, Yili; Li, Xu

    2015-04-01

    Integration of high-risk human papillomavirus (HPV) into the host genome is a key event for cervical carcinogenesis. Different methods have been used to explore the physical states of the HPV genome to reveal the mechanisms for malignant transformation of the infected cells. Consensus has been reached that, although variable portions of the HPV genome are deleted in the integrated HPV sequences, common disruption of the viral E2 gene has been demonstrated in different studies. The head-to-tail concatemers of the full-length HPV16 genome is another typical integration pattern of HPV16, typically found in Caski cell lines, but its prevalence in cervical cancer has never been tested. Here, by introducing a modified PCR, we identified this head-to-tail concatemers of full-length HPV genomes in advanced cervical cancer with HPV16 single positive. Our results show that more than half of the cases contain this integrated head-to-tail concatemers of full-length HPV16 genomes. Further studies in two cervical cell lines, Caski cells and Siha cells, revealed a correlation between the prevalence of the spliced variants of integrated HPV16 sequences and the full-length transcription of the integrated head-to-tail concatemers of the full-length HPV16 genome. Based on these results, we propose that HPV16 integrated into host cells by two mechanisms: one mechanism is shared by other DNA virus and cause integration of the head-to-tail concatemers of the viral genome; another is related to the reverse transcription process, which the integrated HPV sequence is generated by the reverse transcription of the viral mRNA.

  14. Passage Number is a Major Contributor to Genomic Structural Variations in Mouse iPS Cells

    PubMed Central

    Liu, Pengfei; Kaplan, Anna; Yuan, Bo; Hanna, Jacob H.; Lupski, James R.; Reiner, Orly

    2014-01-01

    Emergence of genomic instability is a practical issue in preparing neural stem cells (NSCs) and induced pluripotent stem cells (iPSCs). However, it is still not fully understood what the origins and mechanisms for formation are for the genomic alternations observed. Here, we studied the extent of genomic variation on the scale of individual cells originating from the same animal. We used mouse NSCs grown from embryonic cells and iPSCs generated from embryonic brain cells, B cells or fibroblasts, and performed comparative analysis with cultures of fibroblasts from the same mouse. In the first passage of these cell lines, aneuploidies were only observed for chromosomes 6, 11, 12, 19 and Y, which is overall at a rate lower than previously reported; de novo copy number variations (CNVs) were observed in 4.3% of neural iPSCs, 29% of B cell iPSCs, 10% of fibroblast iPSCs, and 1.3% of neurospheres. In contrast, propagation of these first passage cells to a later passage induced additional aneuploidies and CNVs. Breakpoint sequencing analysis suggested that the majority of the detected CNVs arose by replicative mechanisms. Interestingly, we detected identical de novo CNVs in different single cell colonies that appeared to have arisen independently from each other, which suggests a novel CNV formation mechanism in these cells. Our findings provide insights into mechanisms of CNV formation during reprogramming, and suggest that replicative mechanisms for CNV formation accompany mitotic divisions. PMID:24965231

  15. Linking genomic reorganization to tumor initiation via the giant cell cycle

    PubMed Central

    Niu, N; Zhang, J; Zhang, N; Mercado-Uribe, I; Tao, F; Han, Z; Pathak, S; Multani, A S; Kuang, J; Yao, J; Bast, R C; Sood, A K; Hung, M-C; Liu, J

    2016-01-01

    To investigate the mechanisms underlying our recent paradoxical finding that mitotically incapacitated and genomically unstable polyploid giant cancer cells (PGCCs) are capable of tumor initiation, we labeled ovarian cancer cells with α-tubulin fused to green fluorescent protein, histone-2B fused to red fluorescent protein and FUCCI (fluorescent ubiquitination cell cycle indicator), and tracked the spatial and time-dependent change in spindle and chromosomal dynamics of PGCCs using live-cell fluorescence time-lapse recording. We found that single-dose (500 nm) treatment with paclitaxel paradoxically initiated endoreplication to form PGCCs after massive cell death. The resulting PGCCs continued self-renewal via endoreplication and further divided by nuclear budding or fragmentation; the small daughter nuclei then acquired cytoplasm, split off from the giant mother cells and acquired competency in mitosis. FUCCI showed that PGCCs divided via truncated endoreplication cell cycle (endocycle or endomitosis). Confocal microscopy showed that PGCCs had pronounced nuclear fragmentation and lacked expression of key mitotic proteins. PGCC-derived daughter cells were capable of long-term proliferation and acquired numerous new genome/chromosome alterations demonstrated by spectral karyotyping. These data prompt us to conceptualize a giant cell cycle composed of four distinct but overlapping phases, initiation, self-renewal, termination and stability. The giant cell cycle may represent a fundamental cellular mechanism to initiate genomic reorganization to generate new tumor-initiating cells in response to chemotherapy-induced stress and contributes to disease relapse. PMID:27991913

  16. Optimizing eukaryotic cell hosts for protein production through systems biotechnology and genome-scale modeling.

    PubMed

    Gutierrez, Jahir M; Lewis, Nathan E

    2015-07-01

    Eukaryotic cell lines, including Chinese hamster ovary cells, yeast, and insect cells, are invaluable hosts for the production of many recombinant proteins. With the advent of genomic resources, one can now leverage genome-scale computational modeling of cellular pathways to rationally engineer eukaryotic host cells. Genome-scale models of metabolism include all known biochemical reactions occurring in a specific cell. By describing these mathematically and using tools such as flux balance analysis, the models can simulate cell physiology and provide targets for cell engineering that could lead to enhanced cell viability, titer, and productivity. Here we review examples in which metabolic models in eukaryotic cell cultures have been used to rationally select targets for genetic modification, improve cellular metabolic capabilities, design media supplementation, and interpret high-throughput omics data. As more comprehensive models of metabolism and other cellular processes are developed for eukaryotic cell culture, these will enable further exciting developments in cell line engineering, thus accelerating recombinant protein production and biotechnology in the years to come.

  17. Evaluation of the Cow Rumen Metagenome; Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies(Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    SciTech Connect

    Sczyrba, Alex

    2011-10-13

    DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

  18. Genome-Scale Discovery of Cell Wall Biosynthesis Genes in Populus (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

    ScienceCinema

    Muchero, Wellington [Oak Ridge National Laboratory

    2016-07-12

    Wellington Muchero from Oak Ridge National Laboratory gives a talk titled "Discovery of Cell Wall Biosynthesis Genes in Populus" at the JGI 7th Annual Users Meeting: Genomics of Energy & Environment Meeting on March 22, 2012 in Walnut Creek, California.

  19. Evaluation of the Cow Rumen Metagenome; Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies(Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    ScienceCinema

    Sczyrba, Alex [DOE JGI

    2016-07-12

    DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

  20. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells

    PubMed Central

    Kleinstiver, Benjamin P.; Tsai, Shengdar Q.; Prew, Michelle S.; Nguyen, Nhu T.; Welch, Moira M.; Lopez, Jose M.; McCaw, Zachary R.; Aryee, Martin J.; Joung, J. Keith

    2016-01-01

    The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases1 are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9)2–5. We also report that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells. PMID:27347757

  1. Improved Cell-Penetrating Zinc-Finger Nuclease Proteins for Precision Genome Engineering

    PubMed Central

    Liu, Jia; Gaj, Thomas; Wallen, Mark C; Barbas, Carlos F

    2015-01-01

    Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4+ T cells and 17% in CD34+ hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20–30% in CD4+ T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering. PMID:25756962

  2. Empirical estimation of genome-wide significance thresholds based on the 1000 Genomes Project data set

    PubMed Central

    Kanai, Masahiro; Tanaka, Toshihiro; Okada, Yukinori

    2016-01-01

    To assess the statistical significance of associations between variants and traits, genome-wide association studies (GWAS) should employ an appropriate threshold that accounts for the massive burden of multiple testing in the study. Although most studies in the current literature commonly set a genome-wide significance threshold at the level of P=5.0 × 10−8, the adequacy of this value for respective populations has not been fully investigated. To empirically estimate thresholds for different ancestral populations, we conducted GWAS simulations using the 1000 Genomes Phase 3 data set for Africans (AFR), Europeans (EUR), Admixed Americans (AMR), East Asians (EAS) and South Asians (SAS). The estimated empirical genome-wide significance thresholds were Psig=3.24 × 10−8 (AFR), 9.26 × 10−8 (EUR), 1.83 × 10−7 (AMR), 1.61 × 10−7 (EAS) and 9.46 × 10−8 (SAS). We additionally conducted trans-ethnic meta-analyses across all populations (ALL) and all populations except for AFR (ΔAFR), which yielded Psig=3.25 × 10−8 (ALL) and 4.20 × 10−8 (ΔAFR). Our results indicate that the current threshold (P=5.0 × 10−8) is overly stringent for all ancestral populations except for Africans; however, we should employ a more stringent threshold when conducting a meta-analysis, regardless of the presence of African samples. PMID:27305981

  3. Empirical estimation of genome-wide significance thresholds based on the 1000 Genomes Project data set.

    PubMed

    Kanai, Masahiro; Tanaka, Toshihiro; Okada, Yukinori

    2016-10-01

    To assess the statistical significance of associations between variants and traits, genome-wide association studies (GWAS) should employ an appropriate threshold that accounts for the massive burden of multiple testing in the study. Although most studies in the current literature commonly set a genome-wide significance threshold at the level of P=5.0 × 10(-8), the adequacy of this value for respective populations has not been fully investigated. To empirically estimate thresholds for different ancestral populations, we conducted GWAS simulations using the 1000 Genomes Phase 3 data set for Africans (AFR), Europeans (EUR), Admixed Americans (AMR), East Asians (EAS) and South Asians (SAS). The estimated empirical genome-wide significance thresholds were Psig=3.24 × 10(-8) (AFR), 9.26 × 10(-8) (EUR), 1.83 × 10(-7) (AMR), 1.61 × 10(-7) (EAS) and 9.46 × 10(-8) (SAS). We additionally conducted trans-ethnic meta-analyses across all populations (ALL) and all populations except for AFR (ΔAFR), which yielded Psig=3.25 × 10(-8) (ALL) and 4.20 × 10(-8) (ΔAFR). Our results indicate that the current threshold (P=5.0 × 10(-8)) is overly stringent for all ancestral populations except for Africans; however, we should employ a more stringent threshold when conducting a meta-analysis, regardless of the presence of African samples.

  4. Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype

    PubMed Central

    Cameron Thrash, J; Temperton, Ben; Swan, Brandon K; Landry, Zachary C; Woyke, Tanja; DeLong, Edward F; Stepanauskas, Ramunas; Giovannoni, Stephan J

    2014-01-01

    Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%–86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size. PMID:24451205

  5. Massive multiplication of genome and ribosomes in dormant cells (akinetes) of Aphanizomenon ovalisporum (Cyanobacteria).

    PubMed

    Sukenik, Assaf; Kaplan-Levy, Ruth N; Welch, Jessica Mark; Post, Anton F

    2012-03-01

    Akinetes are dormancy cells commonly found among filamentous cyanobacteria, many of which are toxic and/or nuisance, bloom-forming species. Development of akinetes from vegetative cells is a process that involves morphological and biochemical modifications. Here, we applied a single-cell approach to quantify genome and ribosome content of akinetes and vegetative cells in Aphanizomenon ovalisporum (Cyanobacteria). Vegetative cells of A. ovalisporum were naturally polyploid and contained, on average, eight genome copies per cell. However, the chromosomal content of akinetes increased up to 450 copies, with an average value of 119 genome copies per akinete, 15-fold higher than that in vegetative cells. On the basis of fluorescence in situ hybridization, with a probe targeting 16S rRNA, and detection with confocal laser scanning microscopy, we conclude that ribosomes accumulated in akinetes to a higher level than that found in vegetative cells. We further present evidence that this massive accumulation of nucleic acids in akinetes is likely supported by phosphate supplied from inorganic polyphosphate bodies that were abundantly present in vegetative cells, but notably absent from akinetes. These results are interpreted in the context of cellular investments for proliferation following a long-term dormancy, as the high nucleic acid content would provide the basis for extended survival, rapid resumption of metabolic activity and cell division upon germination.

  6. Wheat germ cell-free protein production system for post-genomic research.

    PubMed

    Madono, Masaki; Sawasaki, Tatsuya; Morishita, Ryo; Endo, Yaeta

    2011-04-30

    Genomic information becomes useful knowledge only when the structures and functions of gene products are understood. In spite of a vast array of analytical tools developed for biological studies in recent years, producing proteins at will is still a bottleneck in post-genomic studies. The cell-free protein production system we developed using wheat embryos has enabled us to produce high quality proteins for genome-wide functional and structural analyses and at the same time circumvent almost all the limitations, such as biohazards and costs, that have hampered conventional cell-free protein synthesis systems. In the present article, we introduce examples of our new wheat germ cell-free protein production system and its application to functional and structural analyses, with the focus on the former.

  7. Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin.

    PubMed

    Kaster, Anne-Kristin; Mayer-Blackwell, Koshlan; Pasarelli, Ben; Spormann, Alfred M

    2014-09-01

    The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3 m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle.

  8. Concurrent Whole-Genome Haplotyping and Copy-Number Profiling of Single Cells

    PubMed Central

    Zamani Esteki, Masoud; Dimitriadou, Eftychia; Mateiu, Ligia; Melotte, Cindy; Van der Aa, Niels; Kumar, Parveen; Das, Rakhi; Theunis, Koen; Cheng, Jiqiu; Legius, Eric; Moreau, Yves; Debrock, Sophie; D’Hooghe, Thomas; Verdyck, Pieter; De Rycke, Martine; Sermon, Karen; Vermeesch, Joris R.; Voet, Thierry

    2015-01-01

    Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones. PMID:25983246

  9. Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells.

    PubMed

    Korge, Sandra; Grudziecki, Astrid; Kramer, Achim

    2015-10-01

    Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms.

  10. Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin

    PubMed Central

    Kaster, Anne-Kristin; Mayer-Blackwell, Koshlan; Pasarelli, Ben; Spormann, Alfred M

    2014-01-01

    The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3 m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle. PMID:24599070

  11. Integrated Genome-Based Studies of Shewanella Ecophysiology

    SciTech Connect

    Zhou, Jizhong; He, Zhili

    2014-04-08

    As a part of the Shewanella Federation project, we have used integrated genomic, proteomic and computational technologies to study various aspects of energy metabolism of two Shewanella strains from a systems-level perspective.

  12. A functional genomic screen in planarians identifies novel regulators of germ cell development.

    PubMed

    Wang, Yuying; Stary, Joel M; Wilhelm, James E; Newmark, Phillip A

    2010-09-15

    Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development. The majority of genes we identified encode conserved RNA-binding proteins, several of which have not been implicated previously in germ cell development. We also show that a germ cell-specific subunit of the conserved transcription factor CCAAT-binding protein/nuclear factor-Y is required for maintaining spermatogonial stem cells. Our results demonstrate that conserved transcriptional and post-transcriptional mechanisms regulate germ cell development in planarians. These findings suggest that studies of planarians will inform our understanding of germ cell biology in higher organisms.

  13. Improved Environmental Genomes via Integration of Metagenomic and Single-Cell Assemblies

    PubMed Central

    Mende, Daniel R.; Aylward, Frank O.; Eppley, John M.; Nielsen, Torben N.; DeLong, Edward F.

    2016-01-01

    Assembling complete or near complete genomes from complex microbial communities remains a significant challenge in metagenomic studies. Recent developments in single cell amplified genomes (SAGs) have enabled the sequencing of individual draft genomes representative of uncultivated microbial populations. SAGs suffer from incomplete and uneven coverage due to artifacts that arise from multiple displacement amplification techniques. Conversely, metagenomic sequence data does not suffer from the same biases as SAGs, and significant improvements have been realized in the recovery of draft genomes from metagenomes. Nevertheless, the inherent genomic complexity of many microbial communities often obfuscates facile generation of population genome assemblies from metagenomic data. Here we describe a new method for metagenomic-guided SAG assembly that leverages the advantages of both methods and significantly improves the completeness of initial SAGs assemblies. We demonstrate that SAG assemblies of two cosmopolitan marine lineages–Marine Group 1 Thaumarchaeota and SAR324 clade bacterioplankton–were substantially improved using this approach. Moreover, the improved assemblies strengthened biological inferences. For example, the improved SAR324 clade genome assembly revealed the presence of many genes in phenylalanine catabolism and flagellar assembly that were absent in the original SAG. PMID:26904016

  14. A sequence-based survey of the complex structural organization of tumor genomes

    SciTech Connect

    Collins, Colin; Raphael, Benjamin J.; Volik, Stanislav; Yu, Peng; Wu, Chunxiao; Huang, Guiqing; Linardopoulou, Elena V.; Trask, Barbara J.; Waldman, Frederic; Costello, Joseph; Pienta, Kenneth J.; Mills, Gordon B.; Bajsarowicz, Krystyna; Kobayashi, Yasuko; Sridharan, Shivaranjani; Paris, Pamela; Tao, Quanzhou; Aerni, Sarah J.; Brown, Raymond P.; Bashir, Ali; Gray, Joe W.; Cheng, Jan-Fang; de Jong, Pieter; Nefedov, Mikhail; Ried, Thomas; Padilla-Nash, Hesed M.; Collins, Colin C.

    2008-04-03

    The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using End Sequencing Profiling (ESP), which relies on paired-end sequencing of cloned tumor genomes. In this study, brain, breast, ovary and prostate tumors along with three breast cancer cell lines were surveyed with ESP yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence in situ hybridization (FISH) confirmed translocations and complex tumor genome structures that include coamplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison of the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms (SNPs) revealed candidate somatic mutations and an elevated rate of novel SNPs in an ovarian tumor. These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than previously appreciated and that genomic fusions including fusion transcripts and proteins may be common, possibly yielding tumor-specific biomarkers and therapeutic targets.

  15. Genome editing and stem cell therapy pave the path for new treatment of sickle-cell disease

    PubMed Central

    Randolph, Mary Katherine

    2015-01-01

    Sickle-cell disease (SCD), also known as sickle-cell anemia, is a hereditary blood disorder characterized by the presence of abnormal hemoglobin, the oxygen-carrying protein found in red blood cells. This devastating hematologic disease affects millions of children worldwide. Currently the only available cure is an allogenic hematopoietic stem cell transplant (HSCT) which is limited by the scarcity of fully-matched donors. SCD is caused by a single nucleotide mutation in the beta-globin gene. Correction of this genetic defect would provide a cure for the disease. Two recent murine studies have provided proof of principle for such a strategy by correcting the mutation in hematopoietic stem cells (HSC) using genome editing techniques. With transformative advances being made in the genome editing field, effective and precise manipulation of cellular genomes is becoming highly feasible. Genome editing techniques in combination with stem cell therapy should provide a safe and curative treatment of various genetic diseases such as SCD. PMID:27358890

  16. The Spindle Assembly Checkpoint Safeguards Genomic Integrity of Skeletal Muscle Satellite Cells.

    PubMed

    Kollu, Swapna; Abou-Khalil, Rana; Shen, Carl; Brack, Andrew S

    2015-06-09

    To ensure accurate genomic segregation, cells evolved the spindle assembly checkpoint (SAC), whose role in adult stem cells remains unknown. Inducible perturbation of a SAC kinase, Mps1, and its downstream effector, Mad2, in skeletal muscle stem cells shows the SAC to be critical for normal muscle growth, repair, and self-renewal of the stem cell pool. SAC-deficient muscle stem cells arrest in G1 phase of the cell cycle with elevated aneuploidy, resisting differentiation even under inductive conditions. p21(CIP1) is responsible for these SAC-deficient phenotypes. Despite aneuploidy's correlation with aging, we find that aged proliferating muscle stem cells display robust SAC activity without elevated aneuploidy. Thus, muscle stem cells have a two-step mechanism to safeguard their genomic integrity. The SAC prevents chromosome missegregation and, if it fails, p21(CIP1)-dependent G1 arrest limits cellular propagation and tissue integration. These mechanisms ensure that muscle stem cells with compromised genomes do not contribute to tissue homeostasis.

  17. Molecular Mechanisms Underlying Genomic Instability in Brca-Deficient Cells

    DTIC Science & Technology

    2012-03-01

    Cre recombi - nase complexed with DNA in a site-specific recombination synapse. Nature 389, 40–46. Helmink, B.A., Tubbs, A.T., Dorsett, Y., Bednarski...therapies to reduce lifetime risk of tumor formation in BRCA1 and potentially BRCA2 carriers. 15. SUBJECT TERMS BRCA1, 53BP1, cancer biology, DNA repair...cell’s ability to maintain genomic stability are systems that monitor and repair DNA double strand breaks (DSBs). The objective of this study is to

  18. Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells.

    PubMed

    Mauro, Maurizio; Caradonna, Fabio; Klein, Catherine B

    2016-03-01

    The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose-dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome-wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long-lasting arsenic-induced genomic instability that manifests in several ways, including aberrant chromosomal effects.

  19. Dysregulation of DNA Methylation Induced by Past Arsenic Treatment Causes Persistent Genomic Instability in Mammalian Cells

    PubMed Central

    Mauro, Maurizio; Caradonna, Fabio; Klein, Catherine B.

    2016-01-01

    The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose-dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome-wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long-lasting arsenic-induced genomic instability that manifests in several ways, including aberrant chromosomal effects. PMID:26581878

  20. RNA exosome regulates AID DNA mutator activity in the B cell genome

    PubMed Central

    Pefanis, Evangelos; Basu, Uttiya

    2015-01-01

    The immunoglobulin diversification processes of somatic hypermutation and class switch recombination critically rely on transcription coupled targeting of AID to Ig loci in activated B lymphocytes. AID catalyzes deamination of cytidine deoxynucleotides on exposed single stranded DNA. In addition to driving immunoglobulin diversity, promiscuous targeting of AID mutagenic activity poses a deleterious threat to genomic stability. Recent genome-wide studies have uncovered pervasive AID activity throughout the B cell genome. It is increasingly apparent that AID activity is frequently targeted to genomic loci undergoing early transcription termination where RNA exosome promotes the resolution of stalled transcription complexes via co-transcriptional RNA degradation mechanisms. Here we review aspects and consequences of eukaryotic transcription that lead to early termination, RNA exosome recruitment, and ultimately targeting of AID mutagenic activity. PMID:26073986

  1. Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

    PubMed

    Watanabe, Kohei; Koga, Hajime; Nakamura, Kodai; Fujita, Akiko; Hattori, Akimasa; Matsuda, Masaru; Koga, Akihiko

    2014-04-01

    DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.

  2. Genomic definition of multiple ex vivo regulatory T cell subphenotypes.

    PubMed

    Feuerer, Markus; Hill, Jonathan A; Kretschmer, Karsten; von Boehmer, Harald; Mathis, Diane; Benoist, Christophe

    2010-03-30

    Regulatory T (Treg) cells that express the Foxp3 transcription factor are essential for lymphoid homeostasis and immune tolerance to self. Other nonimmunological functions of Treg cells, such as controlling metabolic function in adipose tissue, are also emerging. Treg cells originate primarily in the thymus, but can also be elicited from conventional T cells by in vivo exposure to low-dose antigen or homeostatic expansion or by activation in the presence of TGFbeta in vitro. Treg cells are characterized by a distinct transcriptional signature controlled in part, but not solely, by Foxp3. For a better perspective on transcriptional control in Treg cells, we compared gene expression profiles of a broad panel of Treg cells from various origins or anatomical locations. Treg cells generated by different means form different subphenotypes and were identifiable by particular combinations of transcripts, none of which fully encompassed the entire Treg signature. Molecules involved in Treg cell effector function, chemokine receptors, and the transcription factors that control them were differentially represented in these subphenotypes. Treg cells from the gut proved dissimilar to cells elicited by exposure to TGFbeta in vitro, but instead they resembled a CD103(+)Klrg1(+) subphenotype preferentially generated in response to lymphopenia.

  3. Creating new β cells: cellular transmutation by genomic alchemy.

    PubMed

    Moss, Larry G

    2013-03-01

    To address insulin insufficiency, diabetes research has long focused on techniques for replacing insulin-producing β cells. Studies in mice have suggested that, under some conditions, α cells possess the capacity to transdifferentiate into β cells, although the mechanisms that drive this conversion are unclear. In this issue, Bramswig et al. analyzed the methylation states of purified human α, β, and acinar cells and found α cells exhibit intrinsic phenotypic plasticity associated with specific histone methylation profiles. In addition to expanding our understanding of this potential source of β cells, this compendium of carefully generated human gene expression and epigenomic data in islet cell subtypes constitutes a truly valuable resource for the field.

  4. CoryneBase: Corynebacterium Genomic Resources and Analysis Tools at Your Fingertips

    PubMed Central

    Tan, Mui Fern; Jakubovics, Nick S.; Wee, Wei Yee; Mutha, Naresh V. R.; Wong, Guat Jah; Ang, Mia Yang; Yazdi, Amir Hessam; Choo, Siew Woh

    2014-01-01

    Corynebacteria are used for a wide variety of industrial purposes but some species are associated with human diseases. With increasing number of corynebacterial genomes having been sequenced, comparative analysis of these strains may provide better understanding of their biology, phylogeny, virulence and taxonomy that may lead to the discoveries of beneficial industrial strains or contribute to better management of diseases. To facilitate the ongoing research of corynebacteria, a specialized central repository and analysis platform for the corynebacterial research community is needed to host the fast-growing amount of genomic data and facilitate the analysis of these data. Here we present CoryneBase, a genomic database for Corynebacterium with diverse functionality for the analysis of genomes aimed to provide: (1) annotated genome sequences of Corynebacterium where 165,918 coding sequences and 4,180 RNAs can be found in 27 species; (2) access to comprehensive Corynebacterium data through the use of advanced web technologies for interactive web interfaces; and (3) advanced bioinformatic analysis tools consisting of standard BLAST for homology search, VFDB BLAST for sequence homology search against the Virulence Factor Database (VFDB), Pairwise Genome Comparison (PGC) tool for comparative genomic analysis, and a newly designed Pathogenomics Profiling Tool (PathoProT) for comparative pathogenomic analysis. CoryneBase offers the access of a range of Corynebacterium genomic resources as well as analysis tools for comparative genomics and pathogenomics. It is publicly available at http://corynebacterium.um.edu.my/. PMID:24466021

  5. GSP: a web-based platform for designing genome-specific primers in polyploids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The primary goal of this research was to develop a web-based platform named GSP for designing genome-specific primers to distinguish subgenome sequences in the polyploid genome background. GSP uses BLAST to extract homeologous sequences of the subgenomes in the existing databases, performed a multip...

  6. Genome editing of human pluripotent stem cells to generate human cellular disease models.

    PubMed

    Musunuru, Kiran

    2013-07-01

    Disease modeling with human pluripotent stem cells has come into the public spotlight with the awarding of the Nobel Prize in Physiology or Medicine for 2012 to Drs John Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent. This discovery has opened the door for the generation of pluripotent stem cells from individuals with disease and the differentiation of these cells into somatic cell types for the study of disease pathophysiology. The emergence of genome-editing technology over the past few years has made it feasible to generate and investigate human cellular disease models with even greater speed and efficiency. Here, recent technological advances in genome editing, and its utility in human biology and disease studies, are reviewed.

  7. Cell memory-based therapy

    PubMed Central

    Anjamrooz, Seyed Hadi

    2015-01-01

    Current cell therapies, despite all of the progress in this field, still faces major ethical, technical and regulatory hurdles. Because these issues possibly stem from the current, restricted, stereotypical view of cell ultrastructure and function, we must think radically about the nature of the cell. In this regard, the author's theory of the cell memory disc offers ‘memory-based therapy’, which, with the help of immune system rejuvenation, nervous system control and microparticle-based biodrugs, may have substantial therapeutic potential. In addition to its potential value in the study and prevention of premature cell aging, age-related diseases and cell death, memory therapy may improve the treatment of diseases that are currently limited by genetic disorders, risk of tumour formation and the availability and immunocompatibility of tissue transplants. PMID:26256679

  8. Larger Daphnia at lower temperature: a role for cell size and genome configuration?

    PubMed

    Jalal, Marwa; Wojewodzic, Marcin W; Laane, Carl Morten M; Hessen, Dag O

    2013-09-01

    Experiments with Daphnia magna and Daphnia pulex raised at 10 and 20 °C yielded larger adult size at the lower temperature. This must reflect increased cell size, increased cell numbers, or a combination of both. As it is difficult to achieve good estimates on cell size in crustaceans, we, therefore, measured nucleus and genome size using flow cytometry at 10 and 20 °C. DNA was stained with propidium iodide, ethidium bromide, and DAPI. Both nucleus and genome size estimates were elevated at 10 °C compared with 20 °C, suggesting that larger body size at low temperature could partly be accredited to an enlarged nucleus and thus cell size. Confocal microscopy observations confirmed the staining properties of fluorochromes. As differences in nucleotide numbers in response of growth temperature within a life span is unlikely, these results seem accredited to changed DNA-fluorochrome binding properties, presumably reflecting increased DNA condensation at low temperature. This implies that genome size comparisons may be impacted by ambient temperature in ectotherms. It also suggests that temperature-induced structural changes in the genome could affect cell size and for some species even body size.

  9. Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells

    PubMed Central

    Shy, Brian R.; MacDougall, Matthew S.; Clarke, Ryan; Merrill, Bradley J.

    2016-01-01

    CRISPR/Cas9 nucleases have enabled powerful, new genome editing capabilities; however, the preponderance of non-homologous end joining (NHEJ) mediated repair events over homology directed repair (HDR) in most cell types limits the ability to engineer precise changes in mammalian genomes. Here, we increase the efficiency of isolating precise HDR-mediated events in mouse embryonic stem (ES) cells by more than 20-fold through the use of co-incidental insertion (COIN) of independent donor DNA sequences. Analysis of on:off-target frequencies at the Lef1 gene revealed that bi-allelic insertion of a PGK-Neo cassette occurred more frequently than expected. Using various selection cassettes targeting multiple loci, we show that the insertion of a selectable marker at one control site frequently coincided with an insertion at an unlinked, independently targeted site, suggesting enrichment of a sub-population of HDR-proficient cells. When individual cell events were tracked using flow cytometry and fluorescent protein markers, individual cells frequently performed either a homology-dependent insertion event or a homology-independent event, but rarely both types of insertions in a single cell. Thus, when HDR-dependent selection donors are used, COIN enriches for HDR-proficient cells among heterogeneous cell populations. When combined with a self-excising selection cassette, COIN provides highly efficient and scarless genome editing. PMID:27484482

  10. The influence of micronutrients in cell culture: a reflection on viability and genomic stability.

    PubMed

    Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Machado, Miriana; Bordin, Diana Lilian; Bergter, Lothar; Prá, Daniel; Henriques, João Antonio Pêgas

    2013-01-01

    Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5-10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.

  11. Development of a platform for single cell genomics using convex lens-induced confinement.

    PubMed

    Mahshid, Sara; Ahamed, Mohammed Jalal; Berard, Daniel; Amin, Susan; Sladek, Robert; Leslie, Sabrina R; Reisner, Walter

    2015-07-21

    We demonstrate a lab-on-a-chip that combines micro/nano-fabricated features with a Convex Lens-Induced Confinement (CLIC) device for the in situ analysis of single cells. A complete cycle of single cell analysis was achieved that includes: cell trapping, cell isolation, lysis, protein digestion, genomic DNA extraction and on-chip genomic DNA linearization. The ability to dynamically alter the flow-cell dimensions using the CLIC method was coupled with a flow-control mechanism for achieving efficient cell trapping, buffer exchange, and loading of long DNA molecules into nanofluidic arrays. Finite element simulation of fluid flow gives rise to optimized design parameters for overcoming the high hydraulic resistance present in the micro/nano-confinement region. By tuning design parameters such as the pressure gradient and CLIC confinement, an efficient on-chip single cell analysis protocol can be obtained. We demonstrate that we can extract Mbp long genomic DNA molecules from a single human lybphoblastoid cell and stretch these molecules in the nanochannels for optical interrogation.

  12. The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

    PubMed Central

    Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Bordin, Diana Lilian; Prá, Daniel; Pêgas Henriques, João Antonio

    2013-01-01

    Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed. PMID:23781504

  13. Acute high-dose X-radiation-induced genomic changes in A549 cells.

    PubMed

    Muradyan, A; Gilbertz, K; Stabentheiner, S; Klause, S; Madle, H; Meineke, V; Ullmann, R; Scherthan, H

    2011-06-01

    Accidents with ionizing radiation often involve single, acute high-dose exposures that can lead to acute radiation syndrome and late effects such as carcinogenesis. To study such effects at the cellular level, we investigated acute ionizing radiation-induced chromosomal aberrations in A549 adenocarcinoma cells at the genome-wide level by exposing the cells to an acute dose of 6 Gy 240 kV X rays. One sham-irradiated clone and four surviving irradiated clones were recovered by minimal dilution and further expanded and analyzed by chromosome painting and tiling-path array CGH, with the nonirradiated clone 0 serving as the control. Acute X-ray exposure induced specific translocations and changes in modal chromosome number in the four irradiated clones. Array CGH disclosed unique and recurrent genomic changes, predominantly losses, and revealed that the fragile sites FRA3B and FRA16D were preferential regions of genomic alterations in all irradiated clones, which is likely related to radioresistant S-phase progression and genomic stress. Furthermore, clone 4 displayed an increased radiosensitivity at doses >5 Gy. Pairwise comparisons of the gene expression patterns of all irradiated clones to the sham-irradiated clone 0 revealed an enrichment of the Gene Ontology term "M Phase" (P = 6.2 × 10(-7)) in the set of differentially expressed genes of clone 4 but not in those of clones 1-3. Ionizing radiation-induced genomic changes and fragile site expression highlight the capacity of a single acute radiation exposure to affect the genome of exposed cells by inflicting genomic stress.

  14. CyanoBase and RhizoBase: databases of manually curated annotations for cyanobacterial and rhizobial genomes.

    PubMed

    Fujisawa, Takatomo; Okamoto, Shinobu; Katayama, Toshiaki; Nakao, Mitsuteru; Yoshimura, Hidehisa; Kajiya-Kanegae, Hiromi; Yamamoto, Sumiko; Yano, Chiyoko; Yanaka, Yuka; Maita, Hiroko; Kaneko, Takakazu; Tabata, Satoshi; Nakamura, Yasukazu

    2014-01-01

    To understand newly sequenced genomes of closely related species, comprehensively curated reference genome databases are becoming increasingly important. We have extended CyanoBase (http://genome.microbedb.jp/cyanobase), a genome database for cyanobacteria, and newly developed RhizoBase (http://genome.microbedb.jp/rhizobase), a genome database for rhizobia, nitrogen-fixing bacteria associated with leguminous plants. Both databases focus on the representation and reusability of reference genome annotations, which are continuously updated by manual curation. Domain experts have extracted names, products and functions of each gene reported in the literature. To ensure effectiveness of this procedure, we developed the TogoAnnotation system offering a web-based user interface and a uniform storage of annotations for the curators of the CyanoBase and RhizoBase databases. The number of references investigated for CyanoBase increased from 2260 in our previous report to 5285, and for RhizoBase, we perused 1216 references. The results of these intensive annotations are displayed on the GeneView pages of each database. Advanced users can also retrieve this information through the representational state transfer-based web application programming interface in an automated manner.

  15. Genomes-based phylogeny of the genus Xanthomonas

    PubMed Central

    2012-01-01

    Background The genus Xanthomonas comprises several plant pathogenic bacteria affecting a wide range of hosts. Despite the economic, industrial and biological importance of Xanthomonas, the classification and phylogenetic relationships within the genus are still under active debate. Some of the relationships between pathovars and species have not been thoroughly clarified, with old pathovars becoming new species. A change in the genus name has been recently suggested for Xanthomonas albilineans, an early branching species currently located in this genus, but a thorough phylogenomic reconstruction would aid in solving these and other discrepancies in this genus. Results Here we report the results of the genome-wide analysis of DNA sequences from 989 orthologous groups from 17 Xanthomonas spp. genomes available to date, representing all major lineages within the genus. The phylogenetic and computational analyses used in this study have been automated in a Perl package designated Unus, which provides a framework for phylogenomic analyses which can be applied to other datasets at the genomic level. Unus can also be easily incorporated into other phylogenomic pipelines. Conclusions Our phylogeny agrees with previous phylogenetic topologies on the genus, but revealed that the genomes of Xanthomonas citri and Xanthomonas fuscans belong to the same species, and that of Xanthomonas albilineans is basal to the joint clade of Xanthomonas and Xylella fastidiosa. Genome reduction was identified in the species Xanthomonas vasicola in addition to the previously identified reduction in Xanthomonas albilineans. Lateral gene transfer was also observed in two gene clusters. PMID:22443110

  16. Graphite-based photovoltaic cells

    DOEpatents

    Lagally, Max; Liu, Feng

    2010-12-28

    The present invention uses lithographically patterned graphite stacks as the basic building elements of an efficient and economical photovoltaic cell. The basic design of the graphite-based photovoltaic cells includes a plurality of spatially separated graphite stacks, each comprising a plurality of vertically stacked, semiconducting graphene sheets (carbon nanoribbons) bridging electrically conductive contacts.

  17. Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition.

    PubMed

    Ren, Jiangtao; Liu, Xiaojun; Fang, Chongyun; Jiang, Shuguang; June, Carl H; Zhao, Yangbing

    2016-11-04

    Purpose: Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative and potentially improves current chimeric antigen receptor (CAR) T-cell therapy against cancers and infectious diseases.Experimental Design: The CRISPR/Cas9 system has recently emerged as a simple and efficient way for multiplex genome engineering. By combining lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.Results: The CRISPR gene-edited CAR T cells showed potent antitumor activities, both in vitro and in animal models and were as potent as non-gene-edited CAR T cells. In addition, the TCR and HLA class I double deficient T cells had reduced alloreactivity and did not cause graft-versus-host disease. Finally, simultaneous triple genome editing by adding the disruption of PD1 led to enhanced in vivo antitumor activity of the gene-disrupted CAR T cells.Conclusions: Gene-disrupted allogeneic CAR and TCR T cells could provide an alternative as a universal donor to autologous T cells, which carry difficulties and high production costs. Gene-disrupted CAR and TCR T cells with disabled checkpoint molecules may be potent effector cells against cancers and infectious diseases. Clin Cancer Res; 1-12. ©2016 AACR.

  18. CRISPR-Cas9 System as a Versatile Tool for Genome Engineering in Human Cells

    PubMed Central

    Wang, Xuelian; Huang, Xiumin; Fang, Xiuli; Zhang, Youzhong; Wang, Wanpeng

    2016-01-01

    Targeted nucleases are influential instruments for intervening in genome revision with great accuracy. RNA-guided Cas9 nucleases produced from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have noticeably altered the means to modify the genomes of distinct organisms. They can be notably used to facilitate effective genome manipulation in eukaryotic cells by clearly detailing a 20-nt targeting sequence inside its directed RNA. We discuss the most recent advancements in the molecular basis of the type II CRISPR/Cas system and encapsulate applications and elements affecting its use in human cells. We also propose possible applications covering its uses ranging from basic science to implementation in the clinic. PMID:27845770

  19. Genomically Encoded Analog Memory with Precise In vivo DNA Writing in Living Cell Populations

    PubMed Central

    Farzadfard, Fahim; Lu, Timothy K.

    2014-01-01

    Cellular memory is crucial to many natural biological processes and for sophisticated synthetic-biology applications. Existing cellular memories rely on epigenetic switches or recombinases, which are limited in scalability and recording capacity. Here, we use the DNA of living cell populations as genomic ‘tape recorders’ for the analog and distributed recording of long-term event histories. We describe a platform for generating single-stranded DNA (ssDNA) in vivo in response to arbitrary transcriptional signals. When co-expressed with a recombinase, these intracellularly expressed ssDNAs target specific genomic DNA addresses, resulting in precise mutations that accumulate in cell populations as a function of the magnitude and duration of the inputs. This platform could enable long-term cellular recorders for environmental and biomedical applications, biological state machines, and enhanced genome engineering strategies. PMID:25395541

  20. Construction of a phylogenetic tree of photosynthetic prokaryotes based on average similarities of whole genome sequences.

    PubMed

    Satoh, Soichirou; Mimuro, Mamoru; Tanaka, Ayumi

    2013-01-01

    Phylogenetic trees have been constructed for a wide range of organisms using gene sequence information, especially through the identification of orthologous genes that have been vertically inherited. The number of available complete genome sequences is rapidly increasing, and many tools for construction of genome trees based on whole genome sequences have been proposed. However, development of a reasonable method of using complete genome sequences for construction of phylogenetic trees has not been established. We have developed a method for construction of phylogenetic trees based on the average sequence similarities of whole genome sequences. We used this method to examine the phylogeny of 115 photosynthetic prokaryotes, i.e., cyanobacteria, Chlorobi, proteobacteria, Chloroflexi, Firmicutes and nonphotosynthetic organisms including Archaea. Although the bootstrap values for the branching order of phyla were low, probably due to lateral gene transfer and saturated mutation, the obtained tree was largely consistent with the previously reported phylogenetic trees, indicating that this method is a robust alternative to traditional phylogenetic methods.

  1. Evidence of codon usage in the nearest neighbor spacing distribution of bases in bacterial genomes

    NASA Astrophysics Data System (ADS)

    Higareda, M. F.; Geiger, O.; Mendoza, L.; Méndez-Sánchez, R. A.

    2012-02-01

    Statistical analysis of whole genomic sequences usually assumes a homogeneous nucleotide density throughout the genome, an assumption that has been proved incorrect for several organisms since the nucleotide density is only locally homogeneous. To avoid giving a single numerical value to this variable property, we propose the use of spectral statistics, which characterizes the density of nucleotides as a function of its position in the genome. We show that the cumulative density of bases in bacterial genomes can be separated into an average (or secular) plus a fluctuating part. Bacterial genomes can be divided into two groups according to the qualitative description of their secular part: linear and piecewise linear. These two groups of genomes show different properties when their nucleotide spacing distribution is studied. In order to analyze genomes having a variable nucleotide density, statistically, the use of unfolding is necessary, i.e., to get a separation between the secular part and the fluctuations. The unfolding allows an adequate comparison with the statistical properties of other genomes. With this methodology, four genomes were analyzed Burkholderia, Bacillus, Clostridium and Corynebacterium. Interestingly, the nearest neighbor spacing distributions or detrended distance distributions are very similar for species within the same genus but they are very different for species from different genera. This difference can be attributed to the difference in the codon usage.

  2. Reassociation kinetics-based approach for partial genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus

    PubMed Central

    2010-01-01

    Background The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence fiscally and technically problematic. To selectively obtain gene-enriched regions of this tick's genome, Cot filtration was performed, and Cot-filtered DNA was sequenced via 454 FLX pyrosequencing. Results The sequenced Cot-filtered genomic DNA was assembled with an EST-based gene index of 14,586 unique entries where each EST served as a potential "seed" for scaffold formation. The new sequence assembly extended the lengths of 3,913 of the 14,586 gene index entries. Over half of the extensions corresponded to extensions of over 30 amino acids. To survey the repetitive elements in the tick genome, the complete sequences of five BAC clones were determined. Both Class I and II transposable elements were found. Comparison of the BAC and Cot filtration data indicates that Cot filtration was highly successful in filtering repetitive DNA out of the genomic DNA used in 454 sequencing. Conclusion Cot filtration is a very useful strategy to incorporate into genome sequencing projects on organisms with large genome sizes and which contain high percentages of repetitive, difficult to assemble, genomic DNA. Combining the Cot selection approach with 454 sequencing and assembly with a pre-existing EST database as seeds resulted in extensions of 27% of the members of the EST database. PMID:20540747

  3. Combining microarray-based genomic selection (MGS) with the Illumina Genome Analyzer platform to sequence diploid target regions.

    PubMed

    Okou, David T; Locke, Adam E; Steinberg, Karyn M; Hagen, Katie; Athri, Prashanth; Shetty, Amol C; Patel, Viren; Zwick, Michael E

    2009-09-01

    Novel methods of targeted sequencing of unique regions from complex eukaryotic genomes have generated a great deal of excitement, but critical demonstrations of these methods efficacy with respect to diploid genotype calling and experimental variation are lacking. To address this issue, we optimized microarray-based genomic selection (MGS) for use with the Illumina Genome Analyzer (IGA). A set of 202 fragments (304 kb total) contained within a 1.7 Mb genomic region on human chromosome X were MGS/IGA sequenced in ten female HapMap samples generating a total of 2.4 GB of DNA sequence. At a minimum coverage threshold of 5X, 93.9% of all bases and 94.9% of segregating sites were called, while 57.7% of bases (57.4% of segregating sites) were called at a 50X threshold. Data accuracy at known segregating sites was 98.9% at 5X coverage, rising to 99.6% at 50X coverage. Accuracy at homozygous sites was 98.7% at 5X sequence coverage and 99.5% at 50X coverage. Although accuracy at heterozygous sites was modestly lower, it was still over 92% at 5X coverage and increased to nearly 97% at 50X coverage. These data provide the first demonstration that MGS/IGA sequencing can generate the very high quality sequence data necessary for human genetics research. All sequences generated in this study have been deposited in NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra, Accession # SRA007913).

  4. Low Base-Substitution Mutation Rate in the Germline Genome of the Ciliate Tetrahymena thermophil.

    PubMed

    Long, Hongan; Winter, David J; Chang, Allan Y-C; Sung, Way; Wu, Steven H; Balboa, Mariel; Azevedo, Ricardo B R; Cartwright, Reed A; Lynch, Michael; Zufall, Rebecca A

    2016-09-15

    Mutation is the ultimate source of all genetic variation and is, therefore, central to evolutionary change. Previous work on Paramecium tetraurelia found an unusually low germline base-substitution mutation rate in this ciliate. Here, we tested the generality of this result among ciliates using Tetrahymena thermophila. We sequenced the genomes of 10 lines of T. thermophila that had each undergone approximately 1,000 generations of mutation accumulation (MA). We applied an existing mutation-calling pipeline and developed a new probabilistic mutation detection approach that directly models the design of an MA experiment and accommodates the noise introduced by mismapped reads. Our probabilistic mutation-calling method provides a straightforward way of estimating the number of sites at which a mutation could have been called if one was present, providing the denominator for our mutation rate calculations. From these methods, we find that T. thermophila has a germline base-substitution mutation rate of 7.61 × 10 (-)  (12) per-site, per cell division, which is consistent with the low base-substitution mutation rate in P. tetraurelia Over the course of the evolution experiment, genomic exclusion lines derived from the MA lines experienced a fitness decline that cannot be accounted for by germline base-substitution mutations alone, suggesting that other genetic or epigenetic factors must be involved. Because selection can only operate to reduce mutation rates based upon the "visible" mutational load, asexual reproduction with a transcriptionally silent germline may allow ciliates to evolve extremely low germline mutation rates.

  5. Physiological levels of reactive oxygen species are required to maintain genomic stability in stem cells.

    PubMed

    Li, Tao-Sheng; Marbán, Eduardo

    2010-07-01

    Stem cell cytogenetic abnormalities constitute a roadblock to regenerative therapies. We investigated the possibility that reactive oxygen species (ROSs) influence genomic stability in cardiac and embryonic stem cells. Karyotypic abnormalities in primary human cardiac stem cells were suppressed by culture in physiological (5%) oxygen, but addition of antioxidants to the medium unexpectedly increased aneuploidy. Intracellular ROS levels were moderately decreased in physiological oxygen, but dramatically decreased by the addition of high-dose antioxidants. Quantification of DNA damage in cardiac stem cells and in human embryonic stem cells revealed a biphasic dose-dependence: antioxidants suppressed DNA damage at low concentrations, but potentiated such damage at higher concentrations. High-dose antioxidants decreased cellular levels of ATM (ataxia-telangiectasia mutated) and other DNA repair enzymes, providing a potential mechanistic basis for the observed effects. These results indicate that physiological levels of intracellular ROS are required to activate the DNA repair pathway for maintaining genomic stability in stem cells. The concept of an "oxidative optimum" for genomic stability has broad implications for stem cell biology and carcinogenesis.

  6. How cancer cells hijack DNA double-strand break repair pathways to gain genomic instability.

    PubMed

    Jeggo, Penny A; Löbrich, Markus

    2015-10-01

    DNA DSBs (double-strand breaks) are a significant threat to the viability of a normal cell, since they can result in loss of genetic material if mitosis or replication is attempted in their presence. Consequently, evolutionary pressure has resulted in multiple pathways and responses to enable DSBs to be repaired efficiently and faithfully. Cancer cells, which are under pressure to gain genomic instability, have a striking ability to avoid the elegant mechanisms by which normal cells maintain genomic stability. Current models suggest that, in normal cells, DSB repair occurs in a hierarchical manner that promotes rapid and efficient rejoining first, with the utilization of additional steps or pathways of diminished accuracy if rejoining is unsuccessful or delayed. In the present review, we evaluate the fidelity of DSB repair pathways and discuss how cancer cells promote the utilization of less accurate processes. Homologous recombination serves to promote accuracy and stability during replication, providing a battlefield for cancer to gain instability. Non-homologous end-joining, a major DSB repair pathway in mammalian cells, usually operates with high fidelity and only switches to less faithful modes if timely repair fails. The transition step is finely tuned and provides another point of attack during tumour progression. In addition to DSB repair, a DSB signalling response activates processes such as cell cycle checkpoint arrest, which enhance the possibility of accurate DSB repair. We consider the ways by which cancers modify and hijack these processes to gain genomic instability.

  7. Whole-genome sequencing of a malignant granular cell tumor with metabolic response to pazopanib