Revision as of 00:26, 19 May 2015

5/18/2015

Microarray Data analysis Workflow

1. Set browser to send downloads to Desktop
2. Followed the Protocal found on OpenWetWare:

Installing R 3.1.0 and the limma package

The following protocol was developed to normalize GCAT and Ontario DNA microarray chip data from the Dahlquist lab using the R Statistical Software and the limma package (part of the Bioconductor Project).

• The normalization procedure has been verified to work with version 3.1.0 of R released in April 2014 (link to download site) and and version 3.20.1 of the limma package ( direct link to download zipped file) on the Windows 7 platform.
  • Note that using other versions of R or the limma package might give different results.
  • Note also that using the 32-bit versus the 64-bit versions of R 3.1.0 will give different results for the normalization out in the 10^-13 or 10^-14 decimal place. The Dahlquist Lab is standardizing on using the 64-bit version of R.
• To install R for the first time, download and run the installer from the link above, accepting the default installation.
• To use the limma package, unzip the file and place the contents into a folder called "limma" in the library directory of the R program. If you accept the default location, that will be C:\Program Files\R\R-3.1.0\library (this will be different on the computers in S120 since you do not have administrator rights).

Running the Normalization Scripts

• Create a folder on your Desktop to store your files for the microarray analysis procedure.
• Download the zipped file that contains the .gpr files and save it to this folder (or move it if it saved in a different folder).
  • Unzip this file using 7-zip. Right-click on the file and select the menu item, "7-zip > Extract Here".
• Download the GCAT_Targets.csv file and Ontario_Targets_wt-dCIN5-dGLN3-dHAP4-dHMO1-dSWI4-dZAP1-Spar_20150514.csv files and save them to this folder (or move them if they saved to a different folder).
• Download the Ontario_Chip_Within-Array_Normalization_modified_20150514.R script and save (or move) it to this folder.
• Download the Within-Array_Normalization_GCAT_and_Merged_Ontario-GCAT_Between-Chip_Normalization_modified_20150514.R script and save (or move) it to this folder.

Within Array Normalization for the Ontario Chips

• Launch R x64 3.1.0 (make sure you are using the 64-bit version).
• Change the directory to the folder containing the targets file and the GPR files for the Ontario chips by selecting the menu item File > Change dir... and clicking on the appropriate directory. You will need to click on the + sign to drill down to the right directory. Once you have selected it, click OK.
• In R, select the menu item File > Source R code..., and select the Ontario_Chip_Within-Array_Normalization_modified_20150514.R script.
  • You will be prompted by an Open dialog for the Ontario targets file. Select the file Ontario_Targets_wt-dCIN5-dGLN3-dHAP4-dHMO1-dSWI4-dZAP1-Spar_20150514.csv and click Open.
  • Wait while R processes your files.

Within Array Normalization for the GCAT Chips and Between Array Normalization for All Chips

• These instructions assume that you have just completed the Within Array Normalization for the Ontario Chips in the section above.
• In R, select the menu item File > Source R code..., and select the Within-Array_Normalization_GCAT_and_Merged_Ontario-GCAT_Between-Chip_Normalization_modified_20150514.R script.
  • You will be prompted by an Open dialog for the GCAT targets file. Select the file GCAT_Targets.csv and click Open.
  • Wait while R processes your files.
• When the processing has finished, you will find two files called GCAT_and_Ontario_Within_Array_Normalization.csv and GCAT_and_Ontario_Final_Normalized_Data.csv in the same folder.
  • Save these files to LionShare and/or to a flash drive.

Visualizing the Normalized Data

Create MA Plots and Box Plots for the GCAT Chips

Input the following code, line by line, into the main R window. Press the enter key after each block of code.

GCAT.GeneList<-RGG$genes$ID

lg<-log2((RGG$R-RGG$Rb)/(RGG$G-RGG$Gb))

• If you get a message saying "NaNs produced" this is OK, proceed to the next step.

r0<-length(lg[1,])
rx<-tapply(lg[,1],as.factor(GCAT.GeneList),mean)
r1<-length(rx)
MM<-matrix(nrow=r1,ncol=r0)

for(i in 1:r0) {MM[,i]<-tapply(lg[,i],as.factor(GCAT.GeneList),mean)}

MC<-matrix(nrow=r1,ncol=r0)

for(i in 1:r0) {MC[,i]<-dw[i]*MM[,i]}

MCD<-as.data.frame(MC)
colnames(MCD)<-chips
rownames(MCD)<-gcatID

la<-(1/2*log2((RGG$R-RGG$Rb)*(RGG$G-RGG$Gb)))

• If you get these Warning messages, it's OK:

1: In (RGG$R - RGG$Rb) * (RGG$G - RGG$Gb) :

NAs produced by integer overflow

2: NaNs produced

r2<-length(la[1,])
ri<-tapply(la[,1],as.factor(GCAT.GeneList),mean)
r3<-length(ri)
AG<-matrix(nrow=r3,ncol=r2)

for(i in 1:r2) {AG[,i]<-tapply(la[,i],as.factor(GCAT.GeneList),mean)}

par(mfrow=c(3,3))

for(i in 1:r2) {plot(AG[,i],MC[,i],main=chips[i],xlab='A',ylab='M',ylim=c(-5,5),xlim=c(0,15))}
browser()

• Maximize the window in which the graphs have appeared. Save the graphs as a JPEG (File>Save As>JPEG>100% quality...). Once the graphs have been saved, close the window. To continue with the rest of the code, press Enter.
  • To make sure that you save the clearest image, do not scroll in the window because a grey bar will appear if you do so.
• The next set of code is for the generation of the GCAT boxplots for the wild-type data.

x0<-tapply(MAG$A[,1],as.factor(MAG$genes$ID),mean)
y0<-length(MAG$A[1,])
x1<-length(x0)
AAG<-matrix(nrow=x1,ncol=y0)

for(i in 1:y0) {AAG[,i]<-tapply(MAG$A[,i],as.factor(MAG$genes$ID),mean)}

par(mfrow=c(3,3))

for(i in 1:y0) {plot(AAG[,i],MG2[,i],main=chips[i],xlab='A',ylab='M',ylim=c(-5,5),xlim=c(0,15))}
browser()

• Maximize the window in which the graphs have appeared. Save the graphs as a JPEG (File>Save As>JPEG>100% quality...). Once the graphs have been saved, close the window. To continue with the rest of the code, press Enter.

par(mfrow=c(1,3))

boxplot(MCD,main="Before Normalization",ylab='Log Fold Change',ylim=c(-5,5),xaxt='n')

axis(1,at=xy.coords(chips)$x,tick=TRUE,labels=FALSE)

text(xy.coords(chips)$x-1,par('usr')[3]-0.6,labels=chips,srt=45,cex=0.9,xpd=TRUE)

boxplot(MG2,main='After Within Array Normalization',ylab='Log Fold Change',ylim=c(-5,5),xaxt='n')

axis(1,at=xy.coords(chips)$x,labels=FALSE)

text(xy.coords(chips)$x-1,par('usr')[3]-0.6,labels=chips,srt=45,cex=0.9,xpd=TRUE)

boxplot(MAD[,Gtop$MasterList],main='After Between Array Normalization',ylab='Log Fold Change',ylim=c(-5,5),xaxt='n')

axis(1, at=xy.coords(chips)$x,labels=FALSE)

text(xy.coords(chips)$x-1,par('usr')[3]-0.6,labels=chips,srt=45,cex=0.9,xpd=TRUE)

• Maximize the window in which the plots have appeared. You may not want to actually maximize them because you might lose the labels on the x axis, but make them as large as you can. Save the plots as a JPEG (File>Save As>JPEG>100% quality...). Once the graphs have been saved, close the window.

Create MA Plots and Box Plots for the Ontario Chips

Input the following code, line by line, into the main R window. Press the enter key after each block of code.

Ontario.GeneList<-RGO$genes$Name

lr<-log2((RGO$R-RGO$Rb)/(RGO$G-RGO$Gb))

• Warning message: "NaNs produced" is OK.

z0<-length(lr[1,])
v0<-tapply(lr[,1],as.factor(Ontario.GeneList),mean)
z1<-length(v0)
MT<-matrix(nrow=z1,ncol=z0)

for(i in 1:z0) {MT[,i]<-tapply(lr[,i],as.factor(Ontario.GeneList),mean)}

MI<-matrix(nrow=z1,ncol=z0)

for(i in 1:z0) {MI[,i]<-ds[i]*MT[,i]}

MID<-as.data.frame(MI)
colnames(MID)<-headers
rownames(MID)<-ontID

ln<-(1/2*log2((RGO$R-RGO$Rb)*(RGO$G-RGO$Gb)))

• Warning messages are OK:

1: In (RGO$R - RGO$Rb) * (RGO$G - RGO$Gb) :

NAs produced by integer overflow

2: NaNs produced

z2<-length(ln[1,])
zi<-tapply(ln[,1],as.factor(Ontario.GeneList),mean)
z3<-length(zi)
AO<-matrix(nrow=z3,ncol=z2)

for(i in 1:z0) {AO[,i]<-tapply(ln[,i],as.factor(Ontario.GeneList),mean)}

strains<-c('wt','dCIN5','dGLN3','dHAP4','dHMO1','dSWI4','dZAP1','Spar')

• After entering the call browser() below, maximize the window in which the graphs have appeared. Save the graphs as a JPEG (File>Save As>JPEG>100% quality...). Once the graphs have been saved, close the window and press Enter for the next set of graphs to appear.
  • The last graph to appear will be the spar graphs.
  • The graphs generated from this code are the before Ontario chips
• Be sure to save the 9 graphs before moving on to the next step

for (i in 1:length(strains)) {
  st<-strains[i]
  lt<-which(Otargets$Strain %in% st)
  if (st=='wt') {
      par(mfrow=c(3,5))
  } else {
      par(mfrow=c(4,5))
  }
  for (i in lt) {
    plot(AO[,i],MI[,i],main=headers[i],xlab="A",ylab="M",ylim=c(-5,5),xlim=c(0,15))
  }
  browser()
} 

• To continue generating plots, press enter.

j0<-tapply(MAO$A[,1],as.factor(MAO$genes[,5]),mean)
k0<-length(MAO$A[1,])
j1<-length(j0)
AAO<-matrix(nrow=j1,ncol=k0)

for(i in 1:k0) {AAO[,i]<-tapply(MAO$A[,i],as.factor(MAO$genes[,5]),mean)}

• Remember, that after entering the call readline('Press Enter to continue'), maximize the window in which the graphs have appeared. Save the graphs as a JPEG (File>Save As>JPEG>100% quality...). Once the graphs have been saved, close the window and press Enter for the next set of graphs to appear.
  • Again, the last graphs to appear will be the spar graphs.
  • These graphs that are produced are for the after Ontario chips
• Again, be sure to save 9 graphs before moving on to the next part of the code.

for (i in 1:length(strains)) {
  st<-strains[i]
  lt<-which(Otargets$Strain %in% st)
  if (st=='wt') {
      par(mfrow=c(3,5))
  } else {
      par(mfrow=c(4,5))
  }
  for (i in lt) {
    plot(AAO[,i],MD2[,i],main=headers[i],xlab="A",ylab="M",ylim=c(-5,5),xlim=c(0,15))
  }
  browser()
}

• To continue generating plots, press enter.

for (i in 1:length(strains)) {
  par(mfrow=c(1,3))
  st<-strains[i]
  lt<-which(Otargets$Strain %in% st)
  if (st=='wt') {
      xcoord<-xy.coords(lt)$x-1
      fsize<-0.9
  } else {
      xcoord<-xy.coords(lt)$x-1.7
      fsize<-0.8
  }
  boxplot(MID[,lt],main='Before Normalization',ylab='Log Fold Change',ylim=c(-5,5),xaxt='n')
  axis(1,at=xy.coords(lt)$x,labels=FALSE)
  text(xcoord,par('usr')[3]-0.65,labels=headers[lt],srt=45,cex=fsize,xpd=TRUE)
  boxplot(MD2[,lt],main='After Within Array Normalization',ylab='Log Fold Change',ylim=c(-5,5),xaxt='n')
  axis(1,at=xy.coords(lt)$x,labels=FALSE)
  text(xcoord,par('usr')[3]-0.65,labels=headers[lt],srt=45,cex=fsize,xpd=TRUE)
  ft<-Otargets$MasterList[which(Otargets$Strain %in% st)]
  boxplot(MAD[,ft],main='After Between Array Normalization',ylab='Log Fold Change',ylim=c(-5,5),xaxt='n')
  axis(1,at=xy.coords(lt)$x,labels=FALSE)
  text(xcoord,par('usr')[3]-0.65,labels=headers[lt],srt=45,cex=fsize,xpd=TRUE)
  browser()
} 

• To continue generating the box plots, press enter.
  • You will have to save 9 plots before you have completed the procedure. The last box plot is for spar.
• Warnings are OK.
• Zip the files of the plots together and upload to LionShare and/or save to a flash drive.
  • 20151804_Kevin_M_GCATandOntario_Pics.zip

Statistical Analysis

• Added the standard name and the master index for all the terms.
• Saved Compiled_Normalized_Data sheet and made a new sheet called Rounded_Normalized_Data
  • ran computation

=ROUND(Compiled_Normalized_Data!D2,4)

for all data.

• Created Master Sheet, which has all knew data free of any computational functions
  • Copied and pasted numbers with special paste: values
• In Master Sheet:
  • Replaced #VALUE with a blank cell. There were 477 replacements

1. Created a new worksheet and named it"(dgln3_ANOVA)
2. Copied all of the data from the "Master_Sheet" worksheet for your strain and pasted it into the new worksheet.
3. At the top of the first column to the right of Spar_LogFC_t120-4 (FD), five column headers were created of the form dgln3_AvgLogFC_(TIME) where (TIME) is 15, 30, 60, 90, 120.
4. In the cell below the dgln3_AvgLogFC_t15 header, I typed =AVERAGE(
5. highlighted all the data in row 2 associated with dgln3_LogFC_t15 (AU2:AX2), press the closing paren key (shift 0),and press the "enter" key.
6. This cell now contains the average of the log fold change data from the first gene at t=15 minutes.
7. Clicked on this cell and position your cursor at the bottom right corner. Double clicked, and the formula was copied to the entire column of 6188 other genes.
8. Repeated steps (4) through (8) with the t30, t60, t90, and the t120 data.
9. Create the column header dgln3_ss_HO in cell FJ1.
10. In FJ2, I typed =SUMSQ(AU2:BN2)
11. In FK1, create the column headers dgln3_ss_(TIME) as in (3).
12. Make a note of how many data points you have at each time point for your strain.
    • 15:4
    • 30:4
    • 60:4
    • 90:4
    • 120:4
13. In FK2, type =SUMSQ(<range of cells for logFC_t15>)-<number of data points>*<AvgLogFC_t15>^2 and hit enter.
    • The phrase <range of cells for logFC_t15> should be replaced by the data range associated with t15.
    • The phrase <number of data points> should be replaced by the number of data points for that timepoint (either 3, 4, or 5).
    • The phrase <AvgLogFC_t15> should be replaced by the cell number in which you computed the AvgLogFC for t15, and the "^2" squares that value.
    • Actual Computation:=SUMSQ(AU2:AX2)-4*FE2^2
    • Upon completion of this single computation, copy the formula throughout the column.
14. Repeated this computation for the t30 through t120 data points.
15. In FP1, create the column header dgln3_SS_full.
16. In the first row below this header, type =sum(<range of cells containing "ss" for each timepoint>) and hit enter.
    • Actual Computation: =SUM(FK2:FO2)
17. In the next two columns to the right, create the headers dgln3_Fstat and dgln3_p-value.
18. Recall the number of data points from (13): call that total n.
19. In the first cell of the dgln3_Fstat column, type =((n-5)/5)*(dgln3_ss_HO-dgln3_SS_full)/dgln3_SS_full and hit enter.
    • =((20-5)/5)*(FJ2-FP2)/FP2
    • Copy to the whole column.
20. In the first cell below the dgln3_p-value header, type =FDIST(<(dgln3)_Fstat>,5,n-5)

Calculate the Bonferroni and p value Correction

1. Labeled FS1 and FT1 dgln3_Bonferroni_p-value.
2. Type the equation =<dgln3_p-value>*6189, Upon completion of this single computation, copy the formula throughout the column.
3. Replaced any corrected p value that is greater than 1 by the number 1 by typing the following formula into FT2 =IF(r2>1,1,r2). Copy the formula throughout the column.

Calculate the Benjamini & Hochberg p value Correction

1. Insert a new worksheet named "dgln3_B&H".
2. Copy and paste the "MasterIndex", "ID", and "Standard Name" columns from your previous worksheet into the first two columns of the new worksheet.
3. Copied unadjusted p values from ANOVA worksheet and pasted it into Column D.
4. Selected all of columns A, B, C, and D. Sorted by ascending values on Column D. Clicked the sort button from A to Z on the toolbar, sorted by column C, smallest to largest.
5. Typeed the header "Rank" in cell E1. Stretched this down to 6190.
6. Calculated the Benjamini and Hochberg p value correction. Typed dgln3_B-H_p-value in cell F1. Typed the following formula in cell F2: =(D2*6189)/E2 and copied that equation to the entire column.
7. Typed "dgln3_B-H_p-value" into cell G1.
8. Typed the following formula into cell G2: =IF(F2>1,1,F2) Copied that equation to the entire column.
9. Selected columns A through G. Sorted them by your MasterIndex in Column A in ascending order.
10. Copy column G and use Paste special > Paste values to paste it into the next column on the right of your ANOVA sheet.

• Upload the .xlsx file that you have just created to LionShare. Send Dr. Dahlquist an e-mail with the link to the file (e-mail kdahlquist at lmu dot edu).

Sanity Check

1. Click on cell A1 and click on the Data tab. Select the Filter icon (looks like a funnel). Little drop-down arrows should appear at the top of each column. This will enable us to filter the data according to criteria we set.
2. Click on the drop-down arrow on dgln3_p-value. Select "Number Filters". In the window that appears, set a criterion that will filter your data so that the p value has to be less than 0.05.
   • p<0.05=1856 (
   • p<0.01=1007
   • p<0.001=398
   • p<0.0001=121
   • Bonderroni = 20
   • B&H = 889