Msaeedi23 Week 11

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Process for GenMapp analysis

Microarray paper: <http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115243> Genome Paper: <http://www.nature.com/ng/journal/v35/n1/full/ng1227.html>

Week 11

  • For the microarray paper (GenMAPP Users only), include the following:
  • Describe the experimental design of the microarray data, including treatments, number of replicates (biological and/or technical), dye swaps.
  • Determine the sample and data relationships, i.e., which files in the data correspond to which samples in the experimental design.
  • Construct a flow chart that illustrates the above.

Unknown Term Definitions

  • Phosphorelay: A multi-stage process, involving the movement of phophoryl groups by histidine kinases in bacterial signal transduction.

< https://en.wiktionary.org/wiki/phosphorelay>

  • Putative: Believed or deemed to be the case; accepted by supposition rather than from proof.

< https://en.wiktionary.org/wiki/putative>

  • Supernatant: Floating on the surface or over something; the chemistry of a liquid-lying above a sediment or settled precipitate.

< http://dictionary.reference.com/browse/supernatant?s=t>

  • Chemiluminescent: The emission of light as the result of a chemical reaction.

< https://en.wikipedia.org/wiki/Chemiluminescence>

  • Endogenous: Growing or developing from within; originating within.

< http://dictionary.reference.com/browse/endogenous>

  • Non-Permeabilized: Made not permeable, unable to infiltrate a surface.

< https://en.wiktionary.org/wiki/nonpermeabilized>

  • Albeit: Although, even if.

< http://dictionary.reference.com/browse/albeit?s=ts>

  • Attenuated: To render less virulent, as a strain of pathogenic virus or bacterium.

< http://dictionary.reference.com/browse/attenuated?s=t>

  • Modulating: To regulate or adjust to a certain measure or proportion; soften or tone down.

< http://dictionary.reference.com/browse/modulating?s=t>

  • Homodimerization: Any reaction which leads to the formation of a homodimer, a molecule composed of paired identical proteins.

< http://dictionary.reference.com/browse/modulating?s=t>

Article Outline

Write an outline of the article. The length should be a minimum of the equivalent of 2 pages of standard 8 1/2 by 11 inch paper (you can use the "Print Preview" option in your browser to see the length). Your outline can be in any form you choose, but you should utilize the wiki syntax of headers and either numbered or bulleted lists to create it. The text of the outline does not have to be complete sentences, but it should answer the questions listed below and have enough information so that others can follow it. However, your outline should be in YOUR OWN WORDS, not copied straight from the article.

    • What is the importance or significance of this work (i.e., your species)?
    • What were the methods used in the study?
    • Briefly state the result shown in each of the figures and tables.
    • How do the results of this study compare to the results of previous studies (See Discussion).
  • For the microarray paper (GenMAPP Users only), include the following:
    • Describe the experimental design of the microarray data, including treatments, number of replicates (biological and/or technical), dye swaps.
  1. This article linked a recent resurgence of pertussis with an expansion of B. pertussis strains containing a novel allele for the pertussis toxin (ptx) promoter ptxP3 in place of the typical ptxP1 promoter. PtxP3 strains were noted to have better fitness due to higher expression rates of the pertussis toxin. In this experiment, a microarray analysis was conducting comparing several ptxP1 strains to several ptxP3 strains.
    • Treatment Group: PtxP3 strains (n=5)
    • Control Group: PtxP1 strains (n=9)
  2. Three or more biological replicates were conducted for each of the 14 "Bordetella pertussis" strains assessed in this paper, making for a total of 57 unique biological replicates. The data collected for all the replicates within each group was averaged and compared for the final microarray analysis.
    • Replicates, including strain names and the number of biological replicates per strain in parentheses:
      • 1949-B0558 (5)
      • 1967-B1213 (3)
      • 1982-B0689 (4)
      • 1988-B2973 (5)
      • 1995-B0602 (4)
      • 1995-B0607 (4)
      • 1996-B0777 (4)
      • 1999-B1834 (4)
      • 2000-B1878 (4)
      • 2000-B1917 (3)
      • 2007-B3104 (4)
      • 2008-B3183 (5)
      • 2008-B3234 (4)
      • 2008-B3265 (4)
      1. Determine the sample and data relationships, i.e., which files in the data correspond to which samples in the experimental design.
      2. Construct a flow chart that illustrates the above.


DOCUMENT ALL STEPS TAKEN, MANIPULATIONS OF FILES AND ALL.

1. Experimental design-what is the treatment and what is the control

  • Biological vs technical replicants- for vibrio control was the untreated strain, technical replicates = splitting a sample, different from biological replicates. Biological replicates are derived from the original strain. *note how many replicates of each. # of chips(microarrays) (for vibrio it was 9, total number of samples)
      • Dye swaps, cy3 green dye and cy5 red. note how many swaps. Swaps done to avoid dye playing an effect in replication.
        • Draw a diagram to help of how chips were obtained.

2. Sample + data relationship. In raw data there should be one file to each chip. Match file name to sample. raw.zip on arrayexpress has one file for each chip. Sdrf.txt open in excel to show whats in the samples-should tell you all of the samples, their matches, and dye swaps.

3. Compiled raw data file. Include columns for: ID, Sample log2FC, sample log2FC. Compiling raw data files into one spreadsheet to prepare for statistical analysis.

4. Normalization + statistical analysis. Customized for each dataset due to different treatments. Either t-test or one way anova. Also due sanity check, which will be a result in the paper. Compare it to what was written in the paper, corresponding to specific genes. Comparing log-fold changes of specific genes between our data and the paper's.

5. GenMAPP + mappfinder. Run data through database created by groupmates.

6. Deliverables-describing methods, production of tables including the sanity check, etc.

  • In discussion compare what we found vs to what they found.

Mahrad Saeedi

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