microarray-ontol-digest Monday, August 20 2001 Volume 01 : Number 010 ---------------------------------------------------------------------- Date: Fri, 3 Aug 2001 09:46:29 -0400 From: Chris Stoeckert Subject: [microarray-ontol] build ontologies - --Apple-Mail-1851361847-1 Content-Transfer-Encoding: 7bit Content-Type: text/plain; format=flowed; charset=us-ascii Dear Group, This mail has 4 parts. The first is Use Cases/ Scenarios. There are the examples applications of the ontology we are are building. I have listed some that I want to do. Distance metrics and error estimations are for building gene networks. Please submit others so that we can use these to evaluate whether the ontology we build is going to be useful for our purposes. The second part is concepts. These are taken from the OWG web page plus the treatment mail I sent out earlier with changes and additions from Bruce Aronow (U Cincinnati). These are our starting concepts to structure and extend. The third part is a list of tools that can be used to structure these concepts. The list could stand some additions so please feel free to send suggestions. Also indicate whether they cost anything to use. The fourth part is a top level ontology in a taxonomic classification to get us started. Note that everything is a biomaterial description (root). 1. Use Cases/ Scenarios: Return a summary of all experiments that use a specified type of biosource. Group the experiments according to treatment. Return a summary of all experiments done examining effects of a specified treatment Group the experiments according to biosource. Return a summary of all experiments measuring the expression of a specified gene. Indicate when experiments confirm results, provide new information, or conflict. Generate a distance metric for experiment types Generate an error estimation for experimental descriptions Others?? 2. Concepts: Biomaterial: The primary source of the nucleic acid used to generate labelled material for the microarray experiment. Biosource: The primary source of the nucleic acid used to generate labelled material for the microarray experiment. Biosample: The biosource after any treatment. Labeled Extract: The biosample after labeling for detection of the nucleic acids. Organism: The genus and species (and subspecies) of the organism from which the biomaterial is derived from. Biosource provider: The resource (e.g, company, hospital, geographical location) used to obtain or purchase the biomaterial. Biosource donor [Name, Donor ID, Geographic Location, Biomaterial Origin, Consent, Surgical Path #, IRB* Protocol#] Biosource Owner [Name, Address, contact information] Biosource type: The procurement type of the biomaterial (e.g., paraffin section, biopsy) Sex: The gender of the organism or the reproductive organs present on the organism (prior to any modification) that the biomaterial is derived from. Term Definition male The organism contains only the reproductive organ that produces male gametes (spermatozoa). female The organism contains only the reproductive organs that produces female gametes (oocytes). both The organism contains both male and female reproductive organs. none The organism does not have reproductive organs. unknown The reproductive organs of the organism are unknown. Age: The time period elapsed since an identifiable point early in the life of an organism. Examples of the identifiable point include conception, birth, or planting. Initial time point Definition birth The time point at the end of parturition. fertilization The time point at which gametes are joined. May also be used for post-coital measurements. hatching The time point at which the organism leaves the egg. planting The time point at which a seed is planted. Developmental stage: The developmental stage of the organism's life cycle during which the biomaterial was extracted. Organism part: The part of the organism's anatomy from which the biomaterial was derived. Strain or line: Animals or plants that have a single ancestral breeding pair or parent as a result of brother x sister or parent x offspring matings. Genetic Variation: The genetic modification introduced into the organism from which the biomaterial was derived. Examples of genetic variation include specification of a transgene or the gene knocked-out. Individual: Identifier or name of the individual organism from which the biomaterial was derived. Individual genetic characteristics: The genotype of the individual organism from which the biomaterial was derived. Individual genetic characteristics include polymorphisms, disease alleles, and haplotypes. Disease state: The name of the pathology diagnosed in the organism from which the biomaterial was derived. The disease state is normal if no disease has been diagnosed. Targeted cell type: The target cell type is the cell of primary interest. The biomaterial may be derived from a mixed population of cells although only one cell type is of interest. Cell line: The identifier for the immortalized cell line if one was used to derive the biomaterial. Biomaterial preparation: A description of the state and condition of the biomaterial. Time of day when the biomaterial was generated (i.e., sampled). Pathological staging: pre or post mortem at sampling state at start of treatment (age, time of day) physio-chemical composition of the sample: amount of material, number of cells, purity protocol: method used. Environmental or experimental history: A description of the conditions the organism has been exposed to that are not one of the variables under study. culture conditions: A description of the isolated environment used to grow organisms or parts of the organism. atmosphere: The gases and their concentrations used during culture. humidity: The percent humidity. temperature: The temperature during culture. light: The photoperiod and type (e.g., natural, restricted wavelength) of light exposure. nutrients: The food provided to the organism (e.g., chow, fertilizer, DEMM 10%FBS, etc.). medium: The physical state or matrix used to provide nutrients to the organism (e.g., liquid, agar, soil) density range: The concentration range of the organism. contaminant organisms: Organisms present that were not planned as part of the study (e.g., mycoplasma). removal of contaminants: Steps taken to eliminate contaminant organisms. host organism or organism parts: Organisms or organism parts used as a designed part of the culture (e.g., red blood cells, stromal cells). generations: The number of cell divisions if the organism or organism part that is cultured is unicellular otherwise the number of breedings. clinical history: The organism's (i.e., the patient's) medical record. Past medical history Current disease history Clinic treatment history Associated Laboratory values family history: Relevant aspects of genetic preconditions or family members clinical history water: additives and treatments bedding barrier facility pathogen test results: both positive and negative. preservation: seed dormancy, frozen storage Treatment: The manipulation of the biomaterial for the purposes of generating one of the variables under study. somatic modification: The organism has had parts removed, added, or rearranged. genetic modification: The organism has had genes removed, added, or rearranged. starvation: The organism (or organism part) has been deprived of nutrients. infection: The organism (or organism part) has been exposed to a virus or pathogen. behavioral stimulus: The organism is forced to respond to a stimulus with some behavior (e.g., avoidance, obtaining a reward, etc.) agent-based treatment: The treatment is effected by a defined chemical, biological, or physical agent. agent type: chemical (drugs), biological (macromolecule), physical (stress from light, temperature, etc.) agent application in vivo, in vitro, in situ qualitative or quantitative treatment protocol: method of treatment treatment parameters: constant, variable treatment duration: length of treatment 3. Ontology tools: GO-EDIT gene ontology editor: http://www.fruitfly.org/annot/go/editor.html (free) GKB-Editor generic knowledge base editor: http://www.ai.sri.com/~gkb (free) UML: Rational Rose http://www.rational.com/products/rose/index.jsp (not free), Embarcadero http://www.embarcadero.com/products/design/design.htm (not free) Robert Stevens from the Bio-ontologies consortium has added two others: > 1. Protege 2000 is the most widely known and used tool for creating > ontologies and knowledge bases. I believe it is open source. It may be > found at http://protege.stanford.edu/ > 2. OILed (http://img.cs.man.ac.uk/oil) is a little editor for creating > the > OIL and DAML+OIL language. Like GKB and Protege, it has a Frame look and > feel, but the option of reasoning support. Oiled is really a toy tool. > It > is reasonably robust, but not supported in any signficiant fashion. It > has > a little manual, but one would have to read a little about OIL in order > to > use some of the features fully (http://www.ontoknowledge.org/oil). At > http://img.cs.man.ac.uk/stevens/tambis-oil.html) there are some > examples of > the tambis and GO ontologies in OIL (more will appear soon). the > advantange > that OILed has, is that one can create simple DAGS, like GO, all the > way to > full logic models; and migrate from the ormer to the latter. > > My bet is that something like the GO editor will have the shallowest > learning curve and Perhaps OILed the sharpest (due to its > expressiveity). > The balance is what you can get for your effort! Please send pointers to others! I will post these on the web page. 4. Top level ontology: All relationships are ISA (class/ subclass). e.g., biosource is a biomaterial state which is a biomaterial description. Biomaterial description Biomaterial state Biosource Biosample Labeled extract Biomaterial inherent property MGED concept descriptions cell source cell source type sex age Non-MGED concept descriptions organism developmental stage organism part strain or line genetic variation individual individual genetic characteristics disease state targeted cell type cell line clinical information Biomaterial external influence Biomaterial preparation Environmental history Treatment Please feel free to critique/ suggest changes Chris - --Apple-Mail-1851361847-1 Content-Transfer-Encoding: quoted-printable Content-Type: text/enriched; charset=us-ascii Dear Group, This mail has 4 parts. The first is Use Cases/ Scenarios. There are the examples applications of the ontology we are are building. I have listed some that I want to do. Distance metrics and error estimations are for building gene networks. Please submit others so that we can use these to evaluate whether the ontology we build is going to be useful for our purposes. The second part is concepts. These are taken from the OWG web page plus the treatment mail I sent out earlier with changes and additions from Bruce Aronow (U Cincinnati). These are our starting concepts to structure and extend. The third part is a list of tools that can be used to structure these concepts. The list could stand some additions so please feel free to send suggestions. Also indicate whether they cost anything to use. The fourth part is a top level ontology in a taxonomic classification to get us started. Note that everything is a biomaterial description (root). 1. Use Cases/ Scenarios: Return a summary of all experiments that use a specified type of biosource. Group the experiments according to treatment. Return a summary of all experiments done examining effects of a specified treatment Group the experiments according to biosource. Return a summary of all experiments measuring the expression of a specified gene. Indicate when experiments confirm results, provide new = information, or conflict. Generate a distance metric for experiment types Generate an error estimation for experimental descriptions Others?? 2. Concepts: Biomaterial: The primary source of the nucleic acid used to generate labelled material for the microarray experiment. Biosource: The primary source of the nucleic acid used to generate labelled material for the microarray experiment. Biosample: The biosource after any treatment. Labeled Extract: The biosample after labeling for detection of the nucleic acids. Organism: The genus and species (and subspecies) of the organism from which the biomaterial is derived from. Biosource provider: The resource (e.g, company, hospital, geographical location) used to obtain or purchase the biomaterial. Biosource donor [Name, Donor ID, Geographic Location, = Biomaterial Origin, Consent, Surgical Path #, IRB* Protocol#] Biosource Owner [Name, Address, contact information] Biosource type: The procurement type of the biomaterial (e.g., paraffin section, biopsy) Sex: The gender of the organism or the reproductive organs present on the organism (prior to any modification) that the biomaterial is derived from. Term Definition male The organism contains only the reproductive organ that produces male gametes (spermatozoa). female The organism contains only the reproductive organs that produces female gametes (oocytes). both The organism contains both male and female reproductive organs. none The organism does not have reproductive organs. unknown The reproductive organs of the organism are unknown. Age: The time period elapsed since an identifiable point early in the life of an organism. Examples of the identifiable point include conception, birth, or planting. Initial time point Definition birth The time point at the end of parturition. fertilization The time point at which gametes are joined. May also be used for post-coital measurements. hatching The time point at which the organism leaves the egg. planting The time point at which a seed is planted. Developmental stage: The developmental stage of the organism's life cycle during which the biomaterial was extracted. Organism part: The part of the organism's anatomy from which the biomaterial was derived. Strain or line: Animals or plants that have a single ancestral breeding pair or parent as a result of brother x sister or parent x offspring matings. Genetic Variation: The genetic modification introduced into the organism from which the biomaterial was derived. Examples of genetic variation include specification of a transgene or the gene knocked-out. Individual: Identifier or name of the individual organism from which the biomaterial was derived. Individual genetic characteristics: The genotype of the individual organism from which the biomaterial was derived. Individual genetic characteristics include polymorphisms, disease alleles, and haplotypes. Disease state: The name of the pathology diagnosed in the organism from which the biomaterial was derived. The disease state is normal if no disease has been diagnosed. Targeted cell type: The target cell type is the cell of primary interest. The biomaterial may be derived from a mixed population of cells although only one cell type is of interest. Cell line: The identifier for the immortalized cell line if one was used to derive the biomaterial. Biomaterial preparation: A description of the state and condition of the biomaterial. Time of day when the biomaterial was generated (i.e., sampled). Pathological staging: pre or post mortem at sampling state at start of treatment (age, time of day) physio-chemical composition of the sample: amount of material, = number of cells, purity protocol: method used. Environmental or experimental history: A description of the conditions the organism has been exposed to that are not one of the variables under study. culture conditions: A description of the isolated environment = used to grow organisms or parts of the organism. atmosphere: The gases and their concentrations used = during culture. humidity: The percent humidity. temperature: The temperature during culture. light: The photoperiod and type (e.g., natural, = restricted wavelength) of light exposure. nutrients: The food provided to the organism (e.g., = chow, fertilizer, DEMM 10%FBS, etc.). medium: The physical state or matrix used to provide = nutrients to the organism (e.g., liquid, agar, soil) density range: The concentration range of the organism. contaminant organisms: Organisms present that were not = planned as part of the study (e.g., mycoplasma). removal of contaminants: Steps taken to eliminate = contaminant organisms. host organism or organism parts: Organisms or organism = parts used as a designed part of the culture (e.g., red blood cells, stromal cells). generations: The number of cell divisions if the = organism or organism part that is cultured is unicellular otherwise the number of breedings. clinical history: The organism's (i.e., the patient's) medical = record. Past medical history Current disease history Clinic treatment history Associated Laboratory values family history: Relevant aspects of genetic preconditions or = family members clinical history water: additives and treatments bedding barrier facility pathogen test results: both positive and negative. preservation: seed dormancy, frozen storage Treatment: The manipulation of the biomaterial for the purposes of generating one of the variables under study. somatic modification: The organism has had parts = removed, added, or rearranged. genetic modification: The organism has had genes = removed, added, or rearranged. starvation: The organism (or organism part) has been = deprived of nutrients. infection: The organism (or organism part) has been = exposed to a virus or pathogen. behavioral stimulus: The organism is forced to respond = to a stimulus with some behavior (e.g., avoidance, obtaining a reward, etc.) agent-based treatment: The treatment is effected by a = defined chemical, biological, or physical agent. agent type: chemical (drugs), biological = (macromolecule), physical (stress from light, temperature, etc.) agent application in vivo, in vitro, in situ qualitative or quantitative =09= treatment protocol: method of treatment treatment parameters: constant, variable treatment duration: length of treatment 3. Ontology tools: GO-EDIT gene ontology editor: = 1A1A,1A1A,FFFFhttp://www.fruitfly.org/ann= ot/go/editor.html (free) GKB-Editor generic knowledge base editor: = 1A1A,1A1A,FFFFhttp://www.ai.sri.com/~gkb<= /color> (free) UML: Rational Rose = 1A1A,1A1A,FFFFhttp://www.rational.com/pro= ducts/rose/index.jsp (not free), Embarcadero = 1A1A,1A1A,FFFFhttp://www.embarcadero.com/= products/design/design.htm (not free) Robert Stevens from the Bio-ontologies consortium has added two others: 1. Protege 2000 is the most widely known and used tool for creating ontologies and knowledge bases. I believe it is open source. It may be found at = 1A1A,1A1A,FFFFhttp://protege.stanford.edu= / 2. OILed = (1A1A,1A1A,FFFFhttp://img.cs.man.ac.uk/oi= l) is a little editor for creating the OIL and DAML+OIL language. Like GKB and Protege, it has a Frame look and feel, but the option of reasoning support. Oiled is really a toy tool. It is reasonably robust, but not supported in any signficiant fashion. It has a little manual, but one would have to read a little about OIL in order to use some of the features fully = (1A1A,1A1A,FFFFhttp://www.ontoknowledge.o= rg/oil). At = 1A1A,1A1A,FFFFhttp://img.cs.man.ac.uk/ste= vens/tambis-oil.html) there are some examples of the tambis and GO ontologies in OIL (more will appear soon). the advantange that OILed has, is that one can create simple DAGS, like GO, all the way to full logic models; and migrate from the ormer to the latter. My bet is that something like the GO editor will have the shallowest learning curve and Perhaps OILed the sharpest (due to its expressiveity). The balance is what you can get for your effort! Please send pointers to others! I will post these on the web page. 4. Top level ontology: All relationships are ISA (class/ subclass). e.g., biosource is a biomaterial state which is a biomaterial description. Biomaterial description Biomaterial state Biosource Biosample Labeled extract Biomaterial inherent property MGED concept descriptions cell source cell source type sex age Non-MGED concept descriptions organism developmental stage organism part strain or line genetic variation individual individual genetic characteristics disease state targeted cell type cell line clinical information Biomaterial external influence Biomaterial preparation Environmental history Treatment Please feel free to critique/ suggest changes =09 Chris - --Apple-Mail-1851361847-1-- ------------------------------ Date: Fri, 3 Aug 2001 13:27:12 -0700 (PDT) From: Cathy Ball Subject: Re: [microarray-ontol] Update on Bio-Ontologies meeting This message is in MIME format. The first part should be readable text, while the remaining parts are likely unreadable without MIME-aware tools. Send mail to mime@docserver.cac.washington.edu for more info. - --0-1024703220-996870432=:7292 Content-Type: TEXT/PLAIN; charset=X-UNKNOWN Content-Transfer-Encoding: QUOTED-PRINTABLE Hi, As promised at the Bio-Ontologies meeting, I have prepared a set of real-world examples of sample description. I've included sections from the materials and methods or supplemental web sites for published experiments from S. cerevisiae, H. sapiens and C. elegans. As might be expected, the level of detail available for the different studies varies widely (compare the methods for the Perou breast cancer study to the Reinke C. elegans developmental timecourse). The descriptions are at the end of the e-mail and I've attached a text version, as well. Some of the descriptions from the Alizadeh paper might be inaccurate, largely due to the fact that my expertise in immunology has not significantly increased since kindergarten. Chris, I understand that the AFGC folks sent you some sample desccriptions for Arabidopsis experiments already. If you would like to get them again, please let me know. Cheers, Cathy ***************************************************** Catherine Ball, Ph.D. Stanford Microarray Database Stanford University Medical School Department of Genetics Stanford, CA 94305-5120 USA phone: (650)498-6012 fax: (650)723-3016 e-mail: ball@genome.stanford.edu WWW: genome-www.stanford.edu/ ***************************************************** 1) Gasch, A. et al. (2000) Genomic Expression Changes in the Response of Yeast Cells to Environmental Changes. Mol. Biol. Cell 11(12): 4241-4257 Culture of yeast strain S288c was grown in YP media supplemented with 2% weight-to-volume glucose at 25 C until early- log phase. An aliquot of cells was collected for the time=3D0 reference. YPD heated to 49 o C was added until the cell culture reached 37 o C, and the cells were grown at 37 o C. Samples were removed at 5, 10, 15, 20, 30, 40, 50, and 60 minutes, and total RNA was harvested for array analysis. 2) Gasch, A. et al. (2000) Genomic Expression Changes in the Response of Yeast Cells to Environmental Changes. Mol. Biol. Cell 11(12): 4241-4257 Culture of yeast strain S288c was grown in YP media supplemented with 2% weight-to-volume glucose at 25 C until early- log phase. An aliquot of cells was collected for the time=3D0 reference, and the remaining culture was resuspended in an equal volume of media that had been preheated to 37 o C and returned to 37 o C for growth . Samples were removed at 5, 15, 30, 45, and 60 minutes after cell resuspension. 3) Gasch, A. et al. (2000) Genomic Expression Changes in the Response of Yeast Cells to Environmental Changes. Mol. Biol. Cell 11(12): 4241-4257 A 500 mL YPD culture of yeast strain s288c was grown to OD600 0.4. Following the removal of 100 mL as a reference, 100 mL of prewarmed YPD supplemented with H2O2 (Sigma) was added to give a final concentration of 0.30 mM H2O2. Samples were collected at 10, 20, 30, 40, 50, 60, 80, 100, and 120 minutes. Following sample removal, an equal volume of prewarmed YPD supplemented with 0.3 mM H2O2 was added to the culture to maintain the culture volume. In addition, YPD prewarmed to 30 o C and supplemented with H2O2 was dripped into the culture throughout the experiment. The concentration of H2O2 was monitored every three minutes using a horseradish-peroxidase based assay (Green and Hill, 1984) which showed that the concentration of H2O2 was maintained at 0.32 +/- 0.03mM H2O2 over the course of the experiment (data not shown). 4) Perou, C. M., et al. (2000) Molecular portraits of human breast tumours. Nature 406:747-752 Following surgical excision, a human breast tumor sample was rapidly frozen in liquid N2 and then stored at -80 C until RNA was extracted. 5) Alizadeh, A. A., et al. (2000) Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403:503-511 Total germinal centre B cells and centroblasts were purified from human tonsils as described [Liu, Y. -J. & Banchereau, J. in Handbook of Experimental Immunology (eds Weir, D., Blackwell, C., Herzenberg, L. & Herzenberg, L.) 93.1-93.9 (Blackwell Scientific, Oxford, 1996)]. In vitro stimulation of peripheral B cells was done as described [Allman, D. et al. BCL-6 expression during B-cell activation. Blood 87, 5257-5268 (1996)] using anti-IgM antibody, IL-4 and/or CD40 ligand-containing membranes. Purified samples from multiple donors were pooled. 6) Alizadeh, A. A., et al. (2000) Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403:503-511 Human blood B cells were purified from adult apheresis products or cord blood by magnetic enrichment for CD19+ cells (Miltenyi Biotec). Naive CD27+ B cells and memory CD27- blood B cells were isolated by fluorescent cell sorting starting with CD19+ adult peripheral blood B cells [Klein, U., Rajewsky, K. & Kuppers, R. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J. Exp. Med. 188, 1679-1689 (1998), Tangye, S. G., Liu, Y. J., Aversa, G., Phillips, J. H. & de Vries, J. E. Identification of functional human splenic memory B cells by expression of CD148 and CD27. J. Exp. Med. 188, 1691-1703 (1998)]. In vitro stimulation of peripheral B cells was done as described [Allman, D. et al. BCL-6 expression during B-cell activation. Blood 87, 5257-5268 (1996)] using anti-IgM antibody, IL-4 and/or CD40 ligand-containing membranes. 7) Alizadeh, A. A., et al. (2000) Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403:503-511 Magnetic cell sorting was used to purify CD4+, CD45RAhigh T cells from human cord blood or adult peripheral blood and CD4+ thymocytes from human fetal thymus (Milteni Biotec). T cells were stimulated for 2 h with phorbol ester (50 ng ml -1) and ionomycin (1.5 =B5M). 8) Iyer, V., et al. (1999) The transcriptional program in the response of human fibroblasts to serum. Science 283(5398):83-87 A normal human diploid fibroblast cell line derived from foreskin (ATCC CRL 2091) in passage 8 was used in these experiments. The protocol followed for growth arrest and stimulation was essentially that of (16) and (17). Cells were grown to about 60% confluence in 15-cm petri dishes in Dulbecco's minimum essential medium containing glucose (1 g/liter), the antibiotics penicillin and streptomycin, and 10% (by vol) FBS (Hyclone) that had been previously heat inactivated at 56=B0C for 30 min. The cells were then washed three times with the same medium lacking FBS, and low-serum medium (0.1% FBS) was added to the plates. After a 48-hour incubation, the medium was replaced with fresh medium containing 10% FBS. mRNA was isolated from several plates of cells harvested before serum stimulation; this mRNA served as the serum-starved or time-zero reference sample. Cells were harvested from batches of plates at 11 subsequent intervals (15 min, 30 min, 1, 2, 4, 6, 8, 12, 16, 20, and 24 hours) after the addition of serum. mRNA was also isolated from exponentially growing fibroblasts (not subjected to serum starvation). mRNA was isolated with the FastTrack mRNA isolation kit (Invitrogen), which involves lysis of the cells on the plate. The growth medium was removed, and the cells were quickly washed with phosphate-buffered saline at room temperature. The lysis buffer was added to the plate, transferred to tubes, and frozen in liquid nitrogen. Subsequent steps were performed according to the kit manufacturer's protocols. 9) Methods from Kim lab for growing and synchronizing C. elegans (Reinke, et al(2000) A global profile of germline gene expression in C. elegans. Mol Cell 6:605-616) Start by growing 25 medium (100 mm) plates of C. elegans containing a mixture of young and adult worms. When they have nearly starved the plates, wash the worms 3 times with S-basal or M9 solution, pipetting the liquid onto each plate and swirling before pipetting off. You can wash them directly into 500 mL of S-basal in a baffled flask or if you want to quantitate the volume of worms, collect in a 50 mL conical first and spin down. Transfer to a flast and add 3mL E. Coli (amount depends on amount of worms and/or quality of batch of E.Coli but generally speaking start with no more than 1 mL). Shake @20=B0 C on a platform shaker at ~240 rpm. Feed with 1-3 ml E. coli every day. After 4-5 days, separate the live worms from all the debris/dead worms in the culture. This can be done by sucrose floating as follows: 1.Prepare ice cold 0.1 M NaCl and ice cold 60% sucrose. (We like to keep 1-1.5L stocks in the cold room) 2.Spin down the worms in 50mL conicals in a clinical centrifuge (highest or 2nd highest setting; this will take you 2-3 rounds of 4 conicals at a time). Spin time is not that critical, approx. 1 minute is sufficient to yield a soft brown pellet at the bottom of your tubes. 3.Pour off supernatant , resuspend the pellet s in the 0.1 M NaCl and spin to wash, ultimately combining everything into 1 or two tubes depending on your pellet volumes. It is not advisable to have more than 15 mL in each tube. 4.Add NaCl to bring the volume of worms/liquid up to 25 mL in each tube. Mix well to disperse pellet and let sit on ice for > 10minutes. Then add 25mL cold 60% sucrose to each tube, mix, and spin on high for 5 minutes in the clinical centrifuge. The sucrose solution damages and eventually kills the worms so work fast here. Don't let them be exposed to the sucrose for much more than the 5 minutes. 5.After you spin, you should see a brown pellet at the bottom of the tube and a light brown layer of worms floating at the top. Use a broken pasteur pipette tip or a 10mL glass pipette to carefully remove the worms into a new 50 ml conical with approx. 35 mL cold 0.1 M NaCl. 6.Spin the diluted worms for ~1 minute, pour off supernatant and repeat the wash 2-3 more times to get rid of all sucrose. 7.At this point you are ready to collect eggs. To your worm pellet add 2.5 times the volume of hypochlorite solution ( 40 mL 5% bleach, 5 mL 10M NaOH, fill to 100 mL volume with H2O). Mix gently at room temperature for 5 minutes. This will kill the adults and larvae but not the eggs. It should start disintegrating the carcasses as well though usually not completely. Spin 1 minute and decant supernatant. Wash 3-4 times with 0.1 M NaCl, at some point consolidating into 1 tube. Wash the eggs into 100 mL of fresh S. Basal and let shake O/N at 20=B0 C to hatch. The day after you bleached your worms, repeat the sucrose float plate the eggs onto 60 or so 150 mm plates with confluent growth of E. coli (approximately 10-50ul per plate). Plates are then put at 20=B0 C. For L3 staging timecourse, you will want to begin monitoring your worms under the Nomarski scope 32-36 hrs after plating. We usually begin collecting 1/7th of the plates when the Pnp cells are indicative of early L3 stage, that is they are fairly big and look ready to divide for the first time. Time collection will vary depending on the specific experiment you want to do. Harvesting worms You will want to keep a large stock of M9 at 20=B0 C in preparation for harvesting. Doing almost everything at 20=B0 C, pipette M9 onto the plates you want to collect (several mLs per plate) and let shake or swirl by hand for a few seconds/minutes . Then pipette off the liquid with worms into 50 ml conicals. Repeat step again and wash into conicals. Spin worms down briefly in clinical centrifuge and wash with M9 2-3 times to get rid of as much bacteria as possible. In the past we have had trouble with bacteria washing off in flakes and then spinning down with the worms, thereby inflating the real volume yield of worms. For 7 plates we usually see ~1 mL worms. Transfer to 15 mL conical and add 4x volume of trizol reagent. Vortex briefly, flash freeze in liquid nitrogen, thaw at 37=B0 C and repeat sequence once more. 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(see http://www.cbil.upenn.edu/Ontology/Build_Ontology.html) I added figures of relevant sections from MIAME and MAGE for comparison and discussion. Will continue to tweak and add things especially contributions from the group but will start actually working on building the ontology. Hope some of you are too! Chris Chris Stoeckert, Ph.D. Research Associate Professor, Dept. of Genetics Center for Bioinformatics, University of Pennsylvania 418 Guardian Dr., Philadelphia, PA 19104 Ph:215-573-4409 FAX:215-573-3111 ------------------------------ Date: Wed, 8 Aug 2001 12:32:22 +0100 From: Michael Ashburner (Genetics) Subject: Re: [microarray-ontol] new web page for Building an Ontology Does anyone know about the FDA's MedRRA project ? A way of forcing the big pharmas to use a controlled vocab (I think that is it). I can find nothing by an easy web search. Michael Ashburner ------------------------------ Date: Wed, 08 Aug 2001 13:32:14 +0100 From: Helen Parkinson Subject: Re: [microarray-ontol] new web page for Building an Ontology http://www.meddramsso.com/intro/default.htm try this link, seems to be their home page helen "Michael Ashburner (Genetics)" wrote: > > Does anyone know about the FDA's MedRRA project ? A way of forcing > the big pharmas to use a controlled vocab (I think that is it). I > can find nothing by an easy web search. > > Michael Ashburner ------------------------------ Date: Mon, 20 Aug 2001 11:45:59 -0400 From: Chris Stoeckert Subject: [microarray-ontol] Ontology progress and changes - --Apple-Mail-1442731028-6 Content-Transfer-Encoding: 7bit Content-Type: text/plain; format=flowed; charset=us-ascii Dear Group, I've been using the OILed program to structure the top level ontology I mailed you and posted on the OWG site. In doing so, I realized that I would like to make some changes and clarifications. The changes and the new top level ontology are given below. I've also attached the rdfs file generated by OILed to indicate my interpretation of all this. Feel free to argue, point out problems, or suggest additional constraints. Changes: indicated if class (Capitalized and removed spaces between words) added and indicated instances (object, individual) and attributes (slot) changed biosource, biosample, labeled_extract to instances instead of classes removed "MGED concept description" class renamed "Non-MGED concept description" class to BiosourceCVOntology (not really happy with this name) moved Individual class out of BiosourceCVOntology renamed cell source to biosource provider in keeping with the defined concepts on the web page. cell type is now an attribute called biosource type. added tree of "Resource" to indicate people and places you get stuff from. Includes DatabaseEntry for BiosourceCVOntology (based on latest MAGE work) and Donor, Owner for BiosourceProvider. class: BiomaterialDescription class: BiomaterialState instances: biosource, biosample, labeled_extract attribute: has_been_manipulated_by (BiomaterialManipulation) class: BiosourceProperty class: BiosourceProvider attribute: biosource_type (one-of biopsy paraffin_section) attribute: has_donor (Donor) attribute: has_owner (Owner) class: Sex instances: male, female, both_sexes, unknown_sex class: Age attribute: value attribute: unit attribute: initial_time_point (one-of birth, fertilization, hatching, planting) class: Individual class: BiosourceCVOntology // better name for this class? attribute: value attribute: description attribute: ID attribute: has_database_entry class: Organism instance: NCBI_taxonomy class: DevelopmentalStage class: OrganismPart class: StrainOrLine class: GeneticVariation class: IndividualGeneticCharacteristics class: DiseaseState class: TargetedCellType class: CellLine class: ClinicalInformation class: BiomaterialManipulation class: BiomaterialPreparation class: EnvironmentalHistory class: Treatment class: Resource class: DatabaseEntry class: Contact class: Donor class: Owner Cheers, Chris Chris Stoeckert, Ph.D. Research Associate Professor, Dept. of Genetics Center for Bioinformatics, University of Pennsylvania 418 Guardian Dr., Philadelphia, PA 19104 Ph:215-573-4409 FAX:215-573-3111 - --Apple-Mail-1442731028-6 Content-Type: multipart/mixed; boundary=Apple-Mail-912266429-7 - --Apple-Mail-912266429-7 Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii; format=flowed Dear Group, I've been using the OILed program to structure the top level ontology I mailed you and posted on the OWG site. In doing so, I realized that I would like to make some changes and clarifications. The changes and the new top level ontology are given below. I've also attached the rdfs file generated by OILed to indicate my interpretation of all this. Feel free to argue, point out problems, or suggest additional constraints. Changes: indicated if class (Capitalized and removed spaces between words) added and indicated instances (object, individual) and attributes (slot) changed biosource, biosample, labeled_extract to instances instead of classes removed "MGED concept description" class renamed "Non-MGED concept description" class to BiosourceCVOntology (not really happy with this name) moved Individual class out of BiosourceCVOntology renamed cell source to biosource provider in keeping with the defined concepts on the web page. cell type is now an attribute called biosource type. added tree of "Resource" to indicate people and places you get stuff from. Includes DatabaseEntry for BiosourceCVOntology (based on latest MAGE work) and Donor, Owner for BiosourceProvider. class: BiomaterialDescription class: BiomaterialState instances: biosource, biosample, labeled_extract attribute: has_been_manipulated_by (BiomaterialManipulation) class: BiosourceProperty class: BiosourceProvider attribute: biosource_type (one-of biopsy paraffin_section) attribute: has_donor (Donor) attribute: has_owner (Owner) class: Sex instances: male, female, both_sexes, unknown_sex class: Age attribute: value attribute: unit attribute: initial_time_point (one-of birth, fertilization, hatching, planting) class: Individual class: BiosourceCVOntology // better name for this class? attribute: value attribute: description attribute: ID attribute: has_database_entry class: Organism instance: NCBI_taxonomy class: DevelopmentalStage class: OrganismPart class: StrainOrLine class: GeneticVariation class: IndividualGeneticCharacteristics class: DiseaseState class: TargetedCellType class: CellLine class: ClinicalInformation class: BiomaterialManipulation class: BiomaterialPreparation class: EnvironmentalHistory class: Treatment class: Resource class: DatabaseEntry class: Contact class: Donor class: Owner Cheers, Chris Chris Stoeckert, Ph.D. Research Associate Professor, Dept. of Genetics Center for Bioinformatics, University of Pennsylvania 418 Guardian Dr., Philadelphia, PA 19104 Ph:215-573-4409 FAX:215-573-3111 - --Apple-Mail-912266429-7 Content-Disposition: attachment Content-Type: multipart/appledouble; boundary=Apple-Mail-1953012402-8 - --Apple-Mail-1953012402-8 Content-Disposition: attachment; filename="biomaterial.rdfs" Content-Type: application/applefile; name="biomaterial.rdfs" Content-Transfer-Encoding: base64 AAUWBwACAAAAAAAAAAAAAAAAAAAAAAAAAAIAAAAJAAAAMgAAAAoAAAADAAAAPAAAABBURVhUdGV4 dAEAYmlvbWF0ZXJpYWwucmRmcw== - --Apple-Mail-1953012402-8 Content-Disposition: attachment; filename="biomaterial.rdfs" Content-Type: application/text; 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Controlled=20vocabulary=20or=20ontology=20that=20can=20be=20= referred=20to=20in=20order=20to=20describe=20a=20biosource=20= property.=0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09= Properties=20of=20the=20biomaterial=20before=20treated=20in=20= any=20manner=20for=20the=20purposes=20of=20the=20experiment.=0D= =0D=0A=09=0D=0D= =0A=0D=0D=0A=0D=0D=0A=09= The=20resource=20(e.g,=20company,=20hospital,=20geographical=20= location)=20used=20to=20obtain=20or=20purchase=20the=20biomaterial.=20= =0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09The=20identifier=20for=20the=20= immortalized=20cell=20line=20if=20one=20was=20used=20to=20derive=20the=20= biomaterial.=20=0D=0A=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09=0D=0D=0A=0D=0D=0A=0D=0D=0A=09= =0D=0D=0A=0D=0D=0A= =0D=0D=0A=09The=20= developmental=20stage=20of=20the=20organism's=20life=20cycle=20during=20= which=20the=20biomaterial=20was=20extracted.=20=0D=0A=0D=0D=0A= =09=0D=0D=0A= =0D=0D=0A=0D=0D=0A=09The=20= name=20of=20the=20pathology=20diagnosed=20in=20the=20organism=20from=20= which=20the=20biomaterial=20was=20derived.=20The=20disease=20state=20is=20= normal=20if=20no=20disease=20has=20been=20diagnosed.=20=0D=0A= =0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09The=20resource=20(e.g,=20company,=20= hospital,=20geographical=20location)=20used=20to=20obtain=20or=20= purchase=20something.=0D=0D=0A=09=0D=0D=0A=0D=0D=0A=0D=0D=0A=09A=20= description=20of=20the=20conditions=20the=20organism=20has=20been=20= exposed=20to=20that=20are=20not=20one=20of=20the=20variables=20under=20= study.=20=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09The=20= genetic=20modification=20introduced=20into=20the=20organism=20from=20= which=20the=20biomaterial=20was=20derived.=20Examples=20of=20genetic=20= variation=20include=20specification=20of=20a=20transgene=20or=20the=20= gene=20knocked-out.=20=0D=0A=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=0D=0D=0A=09Identifier=20or=20name=20of=20the=20= individual=20organism=20from=20which=20the=20biomaterial=20was=20= derived.=20=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09The=20= genotype=20of=20the=20individual=20organism=20from=20which=20the=20= biomaterial=20was=20derived.=20Individual=20genetic=20characteristics=20= include=20polymorphisms,=20disease=20alleles,=20and=20haplotypes.=20=0D=0A= =0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=0D=0D=0A=09The=20genus=20and=20species=20(and=20= subspecies)=20of=20the=20organism=20from=20which=20the=20biomaterial=20= is=20derived=20from.=20=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09The=20= part=20of=20the=20organism's=20anatomy=20from=20which=20the=20= biomaterial=20was=20derived.=20=0D=0A=0D=0D=0A=09= =0D=0D=0A= =0D=0D=0A=0D=0D=0A=09The=20= individual=20to=20contact=20regarding=20something=20provided=20such=20as=20= a=20biomaterial.=0D=0D=0A=09=0D=0D=0A=0D=0D=0A=0D=0D=0A=09Something=20or=20someone=20you=20get=20= stuff=20from.=20Can=20be=20a=20web=20site,=20organization,=20or=20= individual.=20Stuff=20can=20be=20information=20or=20= biomaterial.=0D=0D=0A=0D=0D=0A=0D=0D=0A=09The=20gender=20of=20the=20organism=20= or=20the=20reproductive=20organs=20present=20on=20the=20organism=20= (prior=20to=20any=20modification)=20that=20the=20biomaterial=20is=20= derived=20from.=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09Animals=20= or=20plants=20that=20have=20a=20single=20ancestral=20breeding=20pair=20= or=20parent=20as=20a=20result=20of=20brother=20x=20sister=20or=20parent=20= x=20offspring=20matings.=20=0D=0A=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09The=20= target=20cell=20type=20is=20the=20cell=20of=20primary=20interest.=20The=20= biomaterial=20may=20be=20derived=20from=20a=20mixed=20population=20of=20= cells=20although=20only=20one=20cell=20type=20is=20of=20interest.=20=0D=0A= =0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09The=20= manipulation=20of=20the=20biomaterial=20for=20the=20purposes=20of=20= generating=20one=20of=20the=20variables=20under=20study.=20=0D= =0D=0A=09=0D= =0D=0A=0D=0D=0A=0D=0D=0A=0D=0D=0A=0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=0D=0D=0A=0D= =0D=0A=0D= =0D=0A=09=0D=0D=0A= =0D=0D=0A=0D=0D=0A=0D=0D=0A=0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09=0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=09= =0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A=0D=0D=0A=0D=0D=0A=09=0D= =0D=0A=09=0D=0D=0A= =0D=0D=0A=0D= =0D=0A=0D=0D=0A=09= =0D=0D=0A=09=0D=0D=0A=0D=0D=0A= =0D=0D=0A= - --Apple-Mail-1953012402-8-- - --Apple-Mail-912266429-7-- - --Apple-Mail-1442731028-6-- ------------------------------ End of microarray-ontol-digest V1 #10 *************************************