Hivanson Week 9

Methods/Results

Strain name: ∆CIN5 strain

Filename: HI_BIOL367_S24_microarray-data_dCIN5.xcls

Number of replicates per strain: 4

Timepoints: 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes

Statistical Analysis Part 1: ANOVA

1. I created a new worksheet and named it "dCIN5_ANOVA"
2. I copied all data from the "Master_Sheet" worksheet and pasted it into dCIN5_ANOVA.
3. I created five column headers of the form dCIN5_AvgLogFC_(TIME) where (TIME) is 15, 30, 60, 90, and 120.
4. In the cell below the dCIN5_AvgLogFC_t15 header, I typed =AVERAGE(
5. Then I highlighted all the data in row 2 associated with t15, pressed the closing paren key, and pressed the "enter" key.
6. I extended this formula down for all genes.
7. I repeated this averaging process with the t30, t60, t90, and the t120 data.
8. In the first empty column to the right of the dCIN5_AvgLogFC_t120 calculation, I created the column header dCIN5_ss_HO.
9. In the first cell below this header, I typed =SUMSQ(
10. I highlighted all the LogFC data in row 2 until the average, pressed the closing paren key, and pressed the "enter" key.
11. In the next empty column to the right of dCIN5_ss_HO, I created the column headers dCIN5_ss_(TIME) as in (3).
12. In the first cell below the header dCIN5_ss_t15, I typed =SUMSQ(<range of cells for logFC_t15>)-COUNTA(<range of cells for logFC_t15>)*<AvgLogFC_t15>^2 and hit enter.
13. I extended this formula down for all genes.
14. I repeated this computation for the t30 through t120 data points. =
15. In the first column to the right of dCIN5_ss_t120, I created the column header dCIN5_SS_full.
16. In the first row below this header, I type =sum(<range of cells containing "ss" for each timepoint>) and hit enter.
17. In the next two columns to the right, I created the headers dCIN5_Fstat and dCIN5_p-value.
18. In the first cell of the dCIN5_Fstat column, I typed =((20-5)/5)*(<dCIN5_ss_HO>-<dCIN5_SS_full>)/<dCIN5_SS_full> and hit enter.
    • I replaced the phrase dCIN5_ss_HO with the cell designation.
    • I replaced the phrase <dCIN5_SS_full> with the cell designation.
    • I copied this to the whole column.
19. In the first cell below the dCIN5_p-value header, I typed =FDIST(<dCIN5_Fstat>,5,20-5)
20. I performed a quick sanity check to see if all of these computations were done correctly.
    • I filtered the dCIN5_p-value column so that the p value has to be less than 0.05.
    • Before further calculation, I undid this filter.

Calculating the Bonferroni and p value Correction

1. I labeled the next two columns to the right with the same label, dCIN5_Bonferroni_p-value.
2. I type the equation =<dCIN5_p-value>*6189, and copied to all genes
3. I replace any corrected p value that is greater than 1 by the number 1 by typing the following formula into the first cell below the second dCIN5_Bonferroni_p-value header: =IF(dCIN5_Bonferroni_p-value>1,1,dCIN5_Bonferroni_p-value), and copied to all genes.

Calculating the Benjamini & Hochberg p value Correction

1. I inserted a new worksheet named "b-h_ANOVA".
2. I copy and paste the "MasterIndex", "ID", and "Standard Name" columns from Master_Sheet_dCIN5 into the first two columns of the new worksheet.
3. I copied my unadjusted p values from your ANOVA worksheet and pasted it into Column D using "paste values."
4. I selected all of columns A, B, C, and D. Sort by ascending values on Column D.
5. I typed the header "Rank" in cell E1 and created a series of numbers in ascending order from 1 to 6189 in this column.
6. To calculate the Benjamini and Hochberg p value correction, I typed dCIN5_B-H_p-value in cell F1. I typed the following formula in cell F2: =(D2*6189)/E2 and pressed enter. I copied that equation to the entire column.
7. I typed "dCIN5-H_p-value" into cell G1.
8. I typed the following formula into cell G2: =IF(F2>1,1,F2) and pressed enter. I copied that equation to the entire column.
9. I selected columns A through G.
10. I sorted them by my Column A MasterIndex in ascending order.
11. I copied column G and used paste values to paste it into the next column on the right of your ANOVA_dCIN5 sheet.

Sanity Check: Number of genes significantly changed

1. In the ANOVA_dCIN5 worksheet, I filtered the unadjusted p value to display only those with a p value of less than 0.05, 0.01, 0.001, and 0.0001.
2. I used =SUBTOTAL(3,A:A) to count the total output, then subtracted 1 to get the number of genes that fit the filter.
3. For the percentage, I used =(100*(<subtotal>-1))/6189). Results as follows:

• How many genes have p < 0.05? and what is the percentage (out of 6189)?
  • 2290 genes; 37.0%

• How many genes have p < 0.01? and what is the percentage (out of 6189)?
  • 1380 genes; 22.3%

• How many genes have p < 0.001? and what is the percentage (out of 6189)?
  • 691 genes; 11.2%

• How many genes have p < 0.0001? and what is the percentage (out of 6189)?
  • 358 genes; 5.8%

1. I repeated the above steps for the Bonferroni-corrected p value of less than 0.05, and the Benjamini and Hochberg-corrected p value of less than 0.05. Results are as follows:

• How many genes are p < 0.05 for the Bonferroni-corrected p value? and what is the percentage (out of 6189)?
  • 151 genes; 2.4%

• How many genes are p < 0.05 for the Benjamini and Hochberg-corrected p value? and what is the percentage (out of 6189)?
  • 1453 genes; 23.5%

Find NSR1 in your dataset. What is its unadjusted, Bonferroni-corrected, and B-H-corrected p values? What is its average Log fold change at each of the timepoints in the experiment?

Unadjusted p value: 6.37625E-08

Bonferroni-corrected p value: 0.000394626

B-H-corrected p value: 2.19237E-05

average Log fold change @ 15 minutes: 4.070025

average Log fold change @ 30 minutes: 3.611475

average Log fold change @ 60 minutes: 4.2985

average Log fold change @ 90 minutes: -2.900925

average Log fold change @ 120 minutes: -0.9315

NSR1 shows increased expression from time 15 minutes through 60 minutes. At 90 minutes, NSR1 expression decreases, and at 120 minutes, the expression of NSR1 remains decreased.

What is IMD3's unadjusted, Bonferroni-corrected, and B-H-corrected p values? What is IMD3's average Log fold change at each of the timepoints in the experiment?

Unadjusted p value: 0.111670609

Bonferroni-corrected p value: 1

B-H-corrected p value: 0.232000469

average Log fold change @ 15 minutes: 1.638433333

average Log fold change @ 30 minutes: -0.100766667

average Log fold change @ 60 minutes: 1.659233333

average Log fold change @ 90 minutes: -0.608333333

average Log fold change @ 120 minutes: -0.168133333

Data & Files

Excel microarray data

p value table slide for ∆CIN5

References

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