Difference between revisions of "Johnllopez Week 8"

Revision as of 03:43, 24 October 2017

Electronic Lab Notebook

Experimental Design and Getting Ready

The strain I used is dSWI4, my timepoints I will be analyzing are t30, t60, t90, and t120. Data was not provided for t15. There were 4 replicates for each of the timepoints.

The first steps I took to complete this assignment were performed in class as I followed along to Dr. Dahlquist's instructions. Note that each time the list below advances 1 number, I performed a save.

1. After initially downloading the Excel document, I went through and deleted all of the columns that did not relate to me and my partner's strain (dsWI4). Then, I went through the data and replaced cells with "NA" with a blank string. There were 3641 replacements.
2. I then renamed the document BIOL367_Fall2017_Dahlquist-microarray-data-master_20171017_JL.

Statistical Analysis Part 1

1. I created a new worksheet named "dSWI4_ANOVA" which would act determine if any of the genes had a significantly different gene expression change han zero at any timepoint.
2. I copied the "MasterIndex", "ID", and "Standard Names" columns from the master sheet, and created 5 column headers in the form of "dSWI4_AvgLogFC_(TIME)", and my time values were 15, 30, 60, 90, and 120.
3. I populated the columns for t30, t60, t90, and t120 by calculating the average of the 4 replicates, using the =AVERAGE() function.

Our total n would be 16 because we are analyzing 4 time points and we have 4 replicates.

1. After letting 16 = n, we applied the two following functions in order to receive our dSWI4_Fstat and dsWI4_p-values: =((n-4)/4)*(Y2-AD2) and =FDIST(AE2,4,n-4).
2. Finally, I filtered through my p-value data to show only p-values less than 0.05. The result was 5475 records found.

Bonferonni and p value Correction

I started this section by creating two new colums with the label "dsWI4_Bonferonni_p-value". Next, I filled the entire first column of that using the following equation: (dSWI4_p-value * 6189) and filled the column AG. Letting that result = AG, I filled the column AE by using the following formula: =IF(AG2>1,1,AG2).

Benjamini and Hochberg p value Correction

To do this, I created a new worksheet to represent the Benjamini and Hochberg p value Correction calculations. I copied the "MasterIndex", "ID", and "Standard Names" columns from the master sheet and the "p-values" sheet from the ANOVA sheet. Then, I sorted these values from smallest to largest by p-value. This was necessary to achieve an index from smallest p-value to largest. Then, I applied the 2 Benjamini and Hochberg p-value correction formulas, which were (D2*6189)/E2 and =IF(F2>1,1,F2). Finally, I put the values in ascending order by MasterIndex, and copied the last column into my ANOVA file.

Sanity Check: Number of genes significantly changed

I then sorted through all of the genes using the following criteria:

1. I saw that 2,802 / 6,189 genes have p <.05, or 45.274%.
2. I saw that 1,842 / 6,189 have p <.01, or 29.762%.
3. I saw that 975 / 6,189 have p < .001, or 15.754%
4. I saw that 512 / 6,189 have p < .0001, or 8.273%.
5. Out of the Bonferonni-corrected p-value, 212 / 6,189 have p < .05, or 3.425%.
6. Out of the Benjamini-Hochberg corrected p-value, 2,076 / 6,189 have p < .05, or 33.543%

Result Comparison

NSR1

• Unaltered P-Value: 1.196 E-7
• Bonferonni-corrected P-Value: 0.0007
• B-H-corrected P-Value: 1
• Average Log Fold Change @ 30: 3.253
• Average Log Fold Change @ 60: 3.565
• Average Log Fold Change @ 90: -3.693
• Average Log Fold Change @ 120: -.084

Given the change in expression from 60 to 90, it would appear that NSR1 changes expression due to cold shock in between this interval.

ADH1

• Unaltered P-Value: .161
• Bonferonni-corrected P-Value: 1
• B-H-corrected P-Value: .554
• Average Log Fold Change @ 30: -.252
• Average Log Fold Change @ 60: -1.126
• Average Log Fold Change @ 90: .144
• Average Log Fold Change @ 120: -.554

Given the change in expression from 60 to 90, it would appear that ADH1 changes expression due to cold shock in between this interval.

My Spreadsheet

File:JL BIOL367 Fall2017 Dahlquist-microarray-data-master 20171017.zipis my document.