Lenaolufson Week 11

Week 11 Notes From Class 11/10/15

(taken from Mahrad's notes page) 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.

Preparation for Journal Club on My Species

10 Unknown Biological Terms

• Murine: Pertaining to or affecting mice or rats.
  • http://medical-dictionary.thefreedictionary.com/murine
• Oligomerization: Formation of oligomers from larger or smaller molecules.
  • http://medical-dictionary.thefreedictionary.com/oligomerization
• Isogenic: Relating to identically identical individuals.
  • http://medical-dictionary.thefreedictionary.com/isogenic
• Ligation: The act of binding or annealing.
  • http://medical-dictionary.thefreedictionary.com/ligation
• Electroporated: A technique in which a brief electric shock is applies to cells; momentary holes open briefly in the plasma membrane, allowing the entry of macromolecules (for example, a way of introducing new DNA into a cell).
  • http://medical-dictionary.thefreedictionary.com/electroporated
• Chemiluminescent: luminescence produced by direct transformation of chemical energy into light energy.
  • http://medical-dictionary.thefreedictionary.com/chemiluminescent
• Endogenous: Produced within or caused by factors within the organism.
  • http://medical-dictionary.thefreedictionary.com/endogenous
• Intranasally: Within the nasal cavity.
  • http://medical-dictionary.thefreedictionary.com/intranasally
• Putative: Considered to exist or have existed.
  • http://www.thefreedictionary.com/putative
• Euthanized: To kill(a person or animal) painlessly, especially to relieve suffering from an incurable illness.
  • http://www.thefreedictionary.com/euthanized

Outline of the article

1. • 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:
     1. Describe the experimental design of the microarray data, including treatments, number of replicates (biological and/or technical), dye swaps.
        • This paper identified polysaccharide (PS) capsules as important virulence determinants for bacterial pathogens and and KpsT as a membrane protein involved in the transport of PS polymers across the cellular envelope in Bordetella pertussis. To determine the impact of PS capsules on the virulence of B. pertussis, a microarray experiment was run testing a ∆KpsT mutant against the wild-type.
        • Treatment Group: ∆KpsT Mutant
        • Control Group: Wild-type (BPSM)
        • Biological replicates: 3 replicates of both the control and treatment groups; Technical replicates: 2 per biological replicate (dye-swapped)
        • Replicate Listings (taken from NCBI GEO):
          • GSM1519507 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 1
          • GSM1519509 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 1 by dye-swap color
          • GSM1519511 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 2
          • GSM1519514 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 2 by dye-swap color
          • GSM1519516 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 3
          • GSM1519519 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 3 by dye-swap color
     2. Determine the sample and data relationships, i.e., which files in the data correspond to which samples in the experimental design.
     3. Construct a flow chart that illustrates the above.