Spikes are at least 1% of total reads

[weshorton]

Summary

The amount of T-cells within a sample is variable. T-cells commonly comprise ~15% of total cells in blood samples, but can range anywhere from 0-15% of total cells in tumor samples. We need to determine the appropriate concentration of spikes to place in each sample, so that the most concentrated blood samples still have spike reads that make up around 1% of total reads.

Significance

It is important for spikes to make up this amount of total reads so that we can be confident in using the variation in their counts to determine the amplification bias for a particular sample. We currently have many spikes with counts of less than 10, making it difficult to determine if the variation between a spike with count 5 is truly half as efficient as a spike with count 10, or if this is just due to random error.

To Do

1. Determine effect of extremely high spike counts within a sample #6
2. Determine primer independence #8

   Approach

3. Control Experiment
   • Samples containing
     • 500 ng DNA (from blood sample)
     • five different spike concentrations (TO DO: what are these?)
   • Five replicates for each spike concentration (25 samples total)
4. Data Analysis and Interpretation
   • Boxplot of spike percent vs. spike concentration
   • Choose spike concentration that gives closest to 1% of reads
5. Relevant plots, tables, and information
   • [Boxplots]

[weshorton]

To Do

1. Make milestone
2. New Issues
3. Analyze data from 160609 spike dilution
   • plots and regression
4. Suggest appropriate spike concentration to use moving forward.