3+Main+Samples+with+lower+secondary+5-1-12

3 Main Samples with lower secondary 5-1-12

experiment performed on 5-4-12

I plan to print 12 tumor cDNA library slides for this experiment 3 for naive sera 3 for tumor sera 3 for SMC1fs sera 3 for 0.1 nM secondary (goat anti-mouse AF647)

I plan to use 0.1 nM secondary instead of 5 nM secondary. I plan to dilute the positive control SMC1fs lysate spots 1/1000 into other lysate if they are not already this way anyway. I plan to still dilute the SMC1fs sera 1:500 I plan to analyze the data by normalizing and subtracting the secondary. I will also make sure that the SMC1fs slide is not blown out. I will decrease the laser power or gain if necessary.

1st scan of files located here: S:\Research\Cancer_Eradication\Users\kwhittem\kwhittem\Raw Data Often Originally on Research Drive\2012\5-5-12 (50% laser power; 50% intensity) This data is also located here S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\Application of sera onto array\3 main low sec 5-10-12\50l-50g An analysis file is here "S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\Application of sera onto array\3 main low sec 5-10-12\50l-50g\p-values.xls"

2nd scans of files located here (more laser powers and gains scanned) S:\Research\Cancer_Eradication\Users\kwhittem\kwhittem\Raw Data Often Originally on Research Drive\2012\5-6-12 This data is also located here "S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\Application of sera onto array\3 main low sec 5-10-12"

Gal file scaled for 10 um "C:\kurt\storage\CIM Research Folder\DR\2012\5-5-12\exple 3k lysate for 10 um.gps"

Here is a summary document for the normalized intensities with secondary subtraction

"C:\kurt\storage\CIM Research Folder\DR\2012\5-11-12\Na‹ve, SMC1fs, and Tumor with Low Secondary 5-11-12.docx"

Some analysis with raw data rather than normalized data C:\kurt\storage\CIM Research Folder\DR\2012\5-14-12\array analysis

Here is a summary document for the raw intensities with no secondary subtraction "C:\kurt\storage\CIM Research Folder\DR\2012\5-14-12\Raw Naive, SMC1fs, and Tumor with Low Secondary 5-14-12.docx"

Potential next steps Maybe I need to remove the e coli lysate antibodies and rerun this sera. For the SMC1fs sample, I could remove GST and e coli lysate antibodies. Then I could reapply this sera to the array.

I would like to compare the tumor spots in this experiment in more detail with the tumor spots from the last 2-23-12 experiment in more detail. I would like to see the correlation with the last experiment as well as if the top binding peptide lists have anything in common (I know that the top 10 have nothing in common between the two experiments already).

Some analysis can be found here: "L:\storage\CIM Research Folder\DR\2012\5-20-12\Comparison of two experiments 5-20-12.xlsx"

The correlation between the two experiments was 0.906. How does this correlation compare to other correlations? > > for the 5-1-12 experiment > 4T1 to 4T1: 0.972 > 4T1 to Naive: (0.963+0.959+0.941)/3 = 0.954 > 4T1 to Secondary: (0.925+0.925+0.902)/3= 0.917 > 4T1 to SMC1fs: (0.64+0.608+0.553)/3 = 0.600
 * Actually not too bad, but not great either. The correlation of tumor to tumor in the 2-23-12 experiment was 0.973, the correlation from tumor to naive was about 0.86, and the correlation from tumor to SMC1fs was about 0.5.

What is the overlap between the top peptide lists in each experiment? (data collected with Genespring) (I was going to write some code to do this, but this became more involved than I anticipated. Since I was trying to write the code in a rush, it wasn't working very well. Nevertheless, the code can still be found here: "L:\storage\CIM Research Folder\DR\2012\5-20-12\code for filtering.txt") Intersection of top 1000 2-23-12 with top 996 5-1-12 has 824 spots overlapping Intersection of top 310 from both experiments has 250 overlapping Intersection of top 56 2-23-12 with top 52 5-1-12 has 47 overlapping

e-mail thread to Kathy about some of these issues https://mail.google.com/mail/u/0/?shva=1#search/kathryn/13752fd48193bcb2

False Discovery Rate analysis

see false discovery analysis

"L:\storage\CIM Research Folder\DR\2012\5-23-12\False discovery rate analysis of several experiments.docx"

I originally tried to look at the false discovery rate of the tumor vs naive with the 25l-50g scan data, but the fdr was 1 for every feature. I would like to see if I obtain the same result with the 50l-50g scan data.

I'll need to align the slides found here. S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\Application of sera onto array\3 main low sec 5-10-12\50l-50g Actually not too bad. The correlation of tumor to tumor in the 2-23-12 experiment was 0.973, the correlation from tumor to naive was about 0.86, and the correlation from tumor to SMC1fs was about 0.5.

I need to compare the differences between the feb experiment and the may experiment and pick a tumor spot to expand out onto a new array. "L:\storage\CIM Research Folder\DR\2012\5-24-12\Comparison of cDNA library array experiments.docx"

I tried scanning the slides one more time at 100% laser 100% gain on 5-25-12. However, analysis of this data did not yield many great p values or false discovery rates between tumor and naive. Analysis can be found here: L:\storage\CIM Research Folder\DR\2012\5-25-12\array analysis

Raw files can be found here:

for the 5-1-12 experiment S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\Application of sera onto array\3 main low sec 5-10-12\100l-100g 5-25-12 4T1 to 4T1: 0.972 4T1 to Naive: (0.963+0.959+0.941)/3 = 0.954 4T1 to Secondary: (0.925+0.925+0.902)/3= 0.917 4T1 to SMC1fs: (0.64+0.608+0.553)/3 = 0.600