Primer competition - Gi

[weshorton]

Summary

Samples have variable initial concentrations of T-Cell DNA, but we spike in a constant amount of synthetic template DNA. How does the variation in T-Cell DNA influence the amplification of spikes? Is this a general phenomenon based on absolute T-Cell concentration, or is it specific to individual primer pairs?

• General (assuming gi):
  • Sample A has twice as much T-Cell DNA as sample B.
  • Spikes in Sample A have half as many counts as spikes in sample B.
• Specific (assuming gi):
  • Sample A has equivalent T-Cell DNA as sample B, but twice as many clonotypes that amplify using V1J1 primer combination (or any primer combination).
  • Spike amplified by V1J1 primer combination (or any combo) has half as many counts in sample A than sample B.
  • Sample A has twice as much T-Cell DNA as sample B, but equivalent amount of clonotypes that amplify using V1J1 primer combination (or any primer combination).
  • Spike amplified by V1J1 primer combination (or any combo) has equivalent counts in sample A and sample B.

    Significance

If gi exists, it must be incorporated into our model - Wiki - Models.

To Do

Determine if gi exists and whether it is general or specific. Use the following approach.

Approach

1. Control Experiment
   • Samples containing:
     • Five replicates of five different concentrations of gDNA (100ng, 200ng, 300ng, 400ng, 500ng)
     • Constant spike concentration of (TO DO: what is this value?)
   • Sample types:
     • Transgenic mouse 1
     • Transgenic mouse 2
     • 50-50 mix of Transgenic mice 1 and 2
     • WT mouse 1
   • 100 total samples
2. Data Analysis and Interpretation
   • Plot total spike count against gDNA concentration
     • Ho: No correlation between initial gDNA concentration and total spike count - no gi
     • Ha: Negative correlation between initial gDNA concentration and total spike count - general gi
   • Plot spike count against clonotype count for each primer pair
     • Ho: No correlation between spike count and clonotype count for primer pairs - no specific gi
     • Ha: Negative correlation between spike count and clonotype count for primer pairs - specific gi
3. Relevant plots, tables, and other information
   • Plots of spike count and clonotype count for primer pairs (for original data, will update with control data)
   • Plots of initial gDNA concentration and total spike count
   • Correlation matrix?

[weshorton]

New To Do (From reorganization mtg)

1. Need to generate spike dilution data (vary gDNA concentration with constant spikes) and analyze
   • plot/regression of dilution factors and spike counts
   • Do for each spike to assess specificity
     1. Utilize existing data
   • Get gDNA concentrations from Dhaarini for particular batch
   • Determine which batch is appropriate
   • Plot gDNA concentration and total spike count with regression