My Computational Journal Summer 2012

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(Week 5: Added entry for June 12, 2012.)
(Week 5: Added entry for June 13, 2012.)
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[[User:Katrina Sherbina|Katrina Sherbina]] 20:59, 12 June 2012 (EDT)
[[User:Katrina Sherbina|Katrina Sherbina]] 20:59, 12 June 2012 (EDT)
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===June 13, 2012===
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Todya, I continued to analyze the mRNA decay experiment microarray data. We decided to do plots of the M values (lnG-lnR) versus time for each gene. However, observing any kind of trend or biase among the arrays this way was already considered a stretch from the onset  but we decided to try anyway. I made the plots specifically for the average PMT data. First, I exported the M values calculated in R and loaded them into MATLAB as a .csv file. Within MATLAB, I constructed a loop that plotted the M values (from the average PMT data) versus time (timepoints in the experiment) for each gene and also a line of best fit through the data points for each gene using the polyfit function. As expected, there was no observable bias across all of the genes. One of the problems may have been that there were no replicates so the lines of best fit probably did not fit the data points well. After sharing these graphs with Fitzpatrick, there is not much else to interpret from this data. We need to talk with Dr. Dahlquist about the experiment and the data more at the next lab  meeting.
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The rest of today was spent continuing to work on the abstract for the mathematical biology conference in July that is due this Friday.
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[[User:Katrina Sherbina|Katrina Sherbina]] 20:47, 13 June 2012 (EDT)

Revision as of 19:47, 13 June 2012

Contents

Week 1

May 14, 2012

Today code for a simple ANOVA and a comparison of two strains was tested to see if the results produced by our adviser could be reproduced. The output without modifying the code for the comparison of two strains using wildtype and dGLN3 for the comparison could be reproduced. A few difficulties have been faced when trying to modify the simple ANOVA code. It was attempted to match the results produced for dGLN3 using the two strain comparison code with the results of the simple ANOVA code with the input specified as the log fold concentrations for the dGLN3 data. After being initially unsuccessful, the code was revisited and looked at more closely. It was found that two matrices in the matrix division had an unequal number of rows (matrix X and matrix Y). The indices (which specify columns of the input file) were modified after which matrix X had the same number of rows as matrix Y. The following code was used:

ind15 = ind15-indx(1)+1;
ind30 = ind30-indx(1)+1;
ind60 = ind60-indx(1)+1;
ind90 = ind90-indx(1)+1;
ind120 = ind120-indx(1)+1;

However, the results from the simple ANOVA for dGLN3 still did not match the results for dGLN3 from the comparison of two strains code.

Katrina Sherbina 21:31, 14 May 2012 (EDT)

May 15, 2012

A separate set of indices were made to designate the rows of the X matrices for the reduced and the full model. This separate set of indices (one for each timpeoint) removed rows of zeros that were previously added when substituting the select 0's for 1's to create the two X matrices. As a result, it was possible to call upon the original indices to extract the log fold changes corresponding to a specific deletion to create the Y matrix. After these corrections, the two strain comparisons were performed between wildtype and each one of the deletion strains.

Further modifications were made to the two strain comparison code to be able to make a comparison of all of the strains simulatenously. To do so, indices were added to designate the columns in the input corresponding to each of the deletion strains. A separate set of indices (one index per timepoint) for each strain as in the case of the two strain comparison. The parameters p were increased to 25 (five timepoints for each deletion strain) and the constraints q were increased to 20 to yield an X and Xh matrices with the appropriate numbers of columns. Changes were made to the out_data(ii,[number]) lines to account for the increase in the number of strains being compared. In the output, the data for each of the timepoints for each of the deletion strains matched the corresponding data in the two strain comparisons.

The next task is figure out how to handle missing data. Since both GCAT and Ontario chips were used, log fold change concentrations are missing for some genes for some timepoints for the wildtype (these cells show up as NaN). One possibility is to modify the X matrices so that they take into account the NaN's for each gene. On this note, a for loop was begun to try to exclude timepoints for a gene for which there is an NaN.

Katrina Sherbina 20:38, 15 May 2012 (EDT)

May 16, 2012

NaN's were removed from the Y matrix for each gene by using the command YY = Y(~isnan(Y(:,1)),1). This truncated the Y matrix from 43 rows to 34 rows. However, in doing so, it was not possible to solve for beta (the solution to the linear system XB=Y). Another method to deal with the NaN's was to replace them with a very large number using the command Y(isnan(Y(:,1)),1)=1e6 in order to be able to recognize the timepoints and flasks for which there were NaN's in the output. However, this messed up the B&H corrections.

The next step will build on trucating the Y matrix by getting rid of the rows with the NaN's altogether. To keep this step, the X and Xh matrix pairs must be formulated so that they are unique to each gene. A possibilty is to use a for loop to define each of the indices for each gene by the columns that do not contain an NaN for that gene. The Y matrix calculations would have to be within this loop.

Katrina Sherbina 21:33, 16 May 2012 (EDT)

May 17, 2012

A for loop was experimented with to generate different X and Xh matrix pairs for each gene to take into account those genes that have an NaN for a a flask for a timepoint in the wildtype. This loop contained a majority of the same coding that was used to set up the indices in the original two strain comparison code. The different lines of code are

for ii=1:n
   I = find(~isnan(a(ii,1:23)));
   ind15 = I(find(I>=1&I<=4));
   ind30 = I(find(I>=5&I<=9));
   ind60 = I(find(I>=10&I<=13));
   ind90 = I(find(I>=14&I<=18));
   ind120 = I(find(I>=19&I<=23));
   indX = find(indx);
end

These new lines of code retain the original column numbers of matrix a (the matrix containing the log fold change data) in the indices. The NaN's were then also removed from the corresponding Y vectors. At first, the loop to compute the Y matrix was put into the loop to compute the X and Xh pairs. However, the code ran for too long at that point. Then the Y loop was separated from the loop to compute the X and Xh matrices. An output was generated. However, for any of the genes that had at least one NaN, the entire row for that gene was blank. In dissecting this new code several problems were found. First, the X matrices for genes that had NaN's were not being computed correctly. Since the indices still had the original column numbers of matrix a, the ones were not put into the X matrix in the correct places. Briefly, cell arrays were looked at as a solution instead of numerical arrays because there can be blank cells in a cell array but numerical arrays do not contain blanks. However, this potential solution proved to complicate the calculations that need to be made.

May 18, 2012

(This entry is being written on May 20th regarding the work that was done on May 18th.)

The first issue that was tackled was the improper formation of the X and Xh matrices. For any of the genes that had at least one NaN, the 1's were put in the wrong positions in the X and Xh matrices. This is because the indices that were used to replace the 0's in the X and Xh matrices with 1 did not start at 1. This was necessary to later exlude the cells that had NaN's in the Y matrix. Rather than getting rid of the method to select the cells that do not have NaN's, the indices that were used to replace the 0's with 1's in the X and Xh matrices were renumbered if indx (the matrix of the indices for each time point) was less than 23 (the maximum number of columns correponding to the wildtype if none of the cells have an NaN for that gene). The following added bit of code was put into the for loop for the X and Xh matrices after the indx, indX, i1, and i2 lines of code:

   if indx<23
       ind15 = find(ind15);
       ind30 = ind30-ind15(end)+1;
       ind60 = ind60-ind30(1)-1;
       ind90 = ind90-ind60(1);
       ind120 = ind120-ind90(1);
   end

However, this may have fixed the X and Xh matrices. However, in the final output, the rows corresponding to the genes with NaN's were still completely blank.

At this point, the wrong direction was taken to find a solution to this. Some experimenting was done to produce X and Xh matrices all of which had 43 rows but some of which had something other than a 0 or 1 in the cell to designate the NaN in the original a matrix. Along this line, the NaN's were kept in the Y matrix. The goal was to be able to somehow ignore the NaN's then when calculating beta and betah.

It was considered to keep the NaN's because it was thought that the problems with the output had to do with some X, Xh, and Y matrices having less than 43 rows while others had 43 rows. Then looking back at how linear systems of equations are solved from what I learned in my linear algebra class, I realized that the number of rows could not be the reason why the some rows of genes in the output were completely blank. Even though the number of rows changed, the number of columns corresponding to the different type points never did. All the X matrices had 10 columns and all the Xh matrices had 5 columns. Therefore, there should be 5 betas being calculated (one for each timepoint) for the wildtype and for dGLN3 for all of the genes regardless of whether or not a gene had NaN's.

The next step in troubleshooting the code will be testing it with just a select number of genes that have NaN's for some timpeoints and flasks.

Katrina Sherbina 23:44, 20 May 2012 (EDT)

Week 2

May 21, 2012

The two strain comparison code was tested with an output that contained only seven genes (six of which had an NaN). At this point, there were no blank rows. To fix the blank rows that appear for genes with NaN's, the computation for the y loop was put in the same loop as the computations for the X and Xh matrices. This ensured that when the betas and betahswere being caluclated that the X and Xh matrices changed accordingly (depending on the gene for which the computation was being done). Previously, the code was set up so that he betas and betahs were calculated with the last X and Xh matrix that was computed in the loop.

Several changes were made to this code to compare all strains (wildtype, dCIN5, dGLN3, dHMO1, and dZAP1). First, indices were created for dCIN5, dGLN3, dHMO1, and dZAP1 (the two strain comparison code only had matrices for the widlytpe and dGLN3). The indices for the deletion strains were constructed for each timepoint similar to how they were constructed for dGLN3 making sure to specifiy the appropriate columns in the original data for each strain.

So that the X and Xh matrices were of the correct size and had 1's and 0's positions in the correct columns and rows, the indices for each timepoint for each strain were adjusted in the for loop.

i3 = ind1202m(end);
   i4 = indx3(1);
   ind153m = ind153-i4+1+i3;
   ind303m = ind303-i4+1+i3;
   ind603m = ind603-i4+1+i3;
   ind903m = ind903-i4+1+i3;
   ind1203m = ind1203-i4+1+i3;
   i5 = ind1203m(end);
   i6 = indx4(1);
   ind154m = ind154-i6+1+i5;
   ind304m = ind304-i6+1+i5;
   ind604m = ind604-i6+1+i5;
   ind904m = ind904-i6+1+i5;
   ind1204m = ind1204-i6+1+i5;
   i7 = ind1204m(end);
   i8 = indx5(1);
   ind155m = ind155-i8+1+i7;
   ind305m = ind305-i8+1+i7;
   ind605m = ind605-i8+1+i7;
   ind905m = ind905-i8+1+i7;
   ind1205m = ind1205-i8+1+i7;

In addition, the the calculations for t, t2, and N were expanded to include all of the indices for all of the strains. The X and Xh matrix for genes with no NaN's had dimensions 103x25 and 103x5, respectively. The X and Xh matrices for the genes with NaN's had dimensions 94x25 and 94x5, respectively.

The coputation for the Y matrix was also altered to include all of the strains:

Y = [a(ii,indx)';a(ii,indx2)';a(ii,indx3)';a(ii,indx4)';a(ii,indx5)'];

The lines of code corresponding to the dimensions of the out_data were also altered to include all of the betas and betahs.

In addition, a few plots from the all strain comparison of the time versus expression genes with both low and high p values were saved. To do so, the if statement for the plots was removed. The save statement used to save each plot generated separately was

saveas(gcf,['Fig' num2str(ii) '.jpg'])

It would be beneficial to change this line of code so that the name of the figure corresponding to the gene for which the expression was being plotted.

Also, an avi file was made of the plots of time versus expression for genes with a p value of less than 0.05 and then those with less than 0.04 (this p value was observed to be the cut off for the B&H corrected p values in the final output for the all strian comparison). The plots for the genes with a p value less than 0.04 have to be redone because a banner that popped up in the figure window was also recorded (it blocks the gene name).

Before the for loop, the following line of code was added

aviobj = avifile('allstrain.avi','fps',15,'compression','Indeo3');

Some fiddling may still need to be done with the number of frames per second.

The following lines of code were added at the end of the for loop (after the if statement for to generate the plots):

   %To get the handles from the figure open. Saves the entire figure window that is open.
   frame = getframe(gcf);
   %Adds the currently open plot to the avi file.
   aviobj = addframe(aviobj,frame);

Right after the for loop, a command line was added to close the avi file:

aviobj = close(aviobj);

The avi file had to be opened in QuickTime player. It would not open in windows media player when either the compression format was Indeo3 or Indeo5.

Katrina Sherbina

May 22, 2012

Some changes were made to the code to write an avi file. However, despite all of the revisions, the avi file was not being made properly. For the first few seconds the plots that showed up for some of the genes looked nothing like they should. To be able to even play the file, the compression had to be set to 'None'. This is what probably caused the file to be 4 GB. They appearance of the strange graphs at the beginning of the movie may be attributed to the large size of the file.

Initially, the p values showing up in the plots in the avi file did not match the p values for the corresponding genes in the output. However, this error was fixed by adjusting the if statement in the loop that creates the plots for genes with a p value of less than 0.05.

    if pval<0.05
       figure(1),plot(t,Y,'bo'),hold on,plot(t,Ym,'r+','LineWidth',3),plot(t,Yh,'k^','LineWidth',3),
        title([b{ii+1,1} '   W = ',num2str(W),' pval = ',num2str(pval)]),...
           xlabel('time, minutes'),ylabel('log fold change'),legend('data','model'),drawnow
       hold off
       nsig = nsig + 1;
       [ii,nsig]
   end

The rest of today was spent dissecting the code we currently have for the deterministic model. The goal is to split the code for the general parameter estimation function into multiple parts, namely, reading the files into matlab, the least squares error function, plotting the graphs, and writing the output.

Katrina Sherbina 20:38, 22 May 2012 (EDT)

May 23, 2012

The lines of code to create an avi file from the plots from the all strains comparison were fixed (thanks to Dr. Fitzpatrick). The changes include the following line of code before the for loop:

figure(1) 
set(gcf, 'DoubleBuffer','on');
yourMovieFileName = ['all_strain_comparison_p_value_less_than_0.05_Cinepak.avi'];

The frame = getframe(gcf) and aviobj = addframe(aviobj,frame) inside the if statement to generate the plots was removed. The aviobj=close(aviobj) line of code immediately after the for loop was replaced with

movie2avi(movieArray,yourMovieFileName,'compression','Cinepak','fps',5,'quality',100);

This time the movie generated with Cinepak compression code be played on Windows Media Player on an XP operating system. The size of this movie was significantly smaller than the size of the previous movie made without compression that could play with Windows Media Player.

There are three different scripts for the statistical analysis in MATLAB:

  • all_strains_compare_movie.m to do a comparison for all strains
  • wildtype_deletion_strain_compare.m to do a comparison between the wildtype and another strain
  • two_deletion_strain_compare.m to do a comparison between two deletion strains

The two strain comparison code was divided into two (the scripts in the last two bullets above) because the for loop designed to remove NaN's is not necessary for the deletion strain data. The principle differences between these two scripts is the calculations to renumber the indices. The following is the part of the script for the wildtype to deletion strain comparisons that contains differences with the other two strain comparison script:

%These indices are changed depending on what deletion strain is being analyzed
ind152  = [33,34,35,36];
ind302  = [37,38,39,40];
ind602  = [41,42,43,44];
ind902  = [45,46,47,48];
ind1202 = [49,50,51,52];
p = 10;
q = 5;
alpha = 0.05;  % significance level for the tests
n152 = length(ind152);
n302 = length(ind302);
n602 = length(ind602);
n902 = length(ind902);
n1202 = length(ind1202);
indx2   = [ind152,ind302,ind602,ind902,ind1202];
n = length(a(:,1));
out_data = zeros(n,12);
nsig = 0;
for ii=1:n
   I = find(~isnan(a(ii,1:23)));
   ind15 = I(find(I>=1&I<=4));
   ind30 = I(find(I>=5&I<=9));
   ind60 = I(find(I>=10&I<=13));
   ind90 = I(find(I>=14&I<=18));
   ind120 = I(find(I>=19&I<=23));
   
   indx    = [ind15,ind30,ind60,ind90,ind120];
   indX    = find(indx);
   
   if length(indx)<23
       ind15 = find(ind15);
       ind30 = ind30 - ind15(end);
       ind60 = ind60 - ind30(1) - 1;
       ind90 = ind90 - ind60(1);
       ind120 = ind120-ind90(1);
   end
     
   n15 = length(ind15);
   n30 = length(ind30);
   n60 = length(ind60);
   n90 = length(ind90);
   n120 = length(ind120);
   
   i1 = indX(end);
   i2 = indx2(1);
   ind152m = ind152-i2+1+i1;
   ind302m = ind302-i2+1+i1;
   ind602m = ind602-i2+1+i1;
   ind902m = ind902-i2+1+i1;
   ind1202m = ind1202-i2+1+i1;
   
   t = [ones(n15,1)*15;ones(n30,1)*30;ones(n60,1)*60;ones(n90,1)*90;ones(n120,1)*120;...
   ones(n152,1)*15;ones(n302,1)*30;ones(n602,1)*60;ones(n902,1)*90;ones(n1202,1)*120];
   t2 = [min(t):5:max(t)]';
   
   N = length(indx)+length(indx2);
   X   = zeros(N,p);
   Xh  = zeros(N,p-q);
   Xh(ind15,1) = 1;
   Xh(ind30,2) = 1;
   Xh(ind60,3) = 1;
   Xh(ind90,4) = 1;
   Xh(ind120,5) = 1;
   Xh(ind152m,1) = 1;
   Xh(ind302m,2) = 1;
   Xh(ind602m,3) = 1;
   Xh(ind902m,4) = 1;
   Xh(ind1202m,5) = 1;
   X(ind15,1) = 1;
   X(ind30,2) = 1;
   X(ind60,3) = 1;
   X(ind90,4) = 1;
   X(ind120,5) = 1;
   X(ind152m,6) = 1;
   X(ind302m,7) = 1;
   X(ind602m,8) = 1;
   X(ind902m,9) = 1;
   X(ind1202m,10) = 1;
end

The calculations for the Y matrices, beta's, betah's, fstats, pvals, and plots are within the for loop. These are the same calculations in the deletion strain to deletion strain comparison script. These caluclations are the only ones in the for loop for the deletion strain to deletion strain comparison script. The following is a part of the deletion strain to deletion strain comparison script that contains differences in comparison to the previous script.

%These indices are changed depending on what deletion strain is being analyzed
ind15  = [91,92,93,94];
ind30  = [95,96,97,98];
ind60  = [99,100,101,102];
ind90  = [103,104,105,106];
ind120 = [107,108,109,110];
ind152  = [120,121,122,123];
ind302  = [124,125,126,127];
ind602  = [128,129,130,131];
ind902  = [132,133,134,135];
ind1202 = [136,137,138,139];
p = 10;
q = 5;
alpha = 0.05;  % significance level for the tests
n15 = length(ind15);
n30 = length(ind30);
n60 = length(ind60);
n90 = length(ind90);
n120 = length(ind120);
n152 = length(ind152);
n302 = length(ind302);
n602 = length(ind602);
n902 = length(ind902);
n1202 = length(ind1202);
indx    = [ind15,ind30,ind60,ind90,ind120];
indX    = find(indx);
indx2   = [ind152,ind302,ind602,ind902,ind1202];
i0 = ind15(1);
i1 = indX(end);
i2 = indx2(1);
ind15m = find(ind15);
ind30m = ind30-i0+1;
ind60m = ind60-i0+1;
ind90m = ind90-i0+1;
ind120m = ind120-i0+1;
ind152m = ind152 -i2+1+i1;
ind302m = ind302 -i2+1+i1;
ind602m = ind602 -i2+1+i1;
ind902m = ind902 -i2+1+i1;
ind1202m = ind1202 -i2+1+i1;
t = [ones(n15,1)*15;ones(n30,1)*30;ones(n60,1)*60;ones(n90,1)*90;ones(n120,1)*120;...
   ones(n152,1)*15;ones(n302,1)*30;ones(n602,1)*60;ones(n902,1)*90;ones(n1202,1)*120];
t2 = [min(t):5:max(t)]';
N = length(indx)+length(indx2);
X   = zeros(N,p);
Xh  = zeros(N,p-q);
Xh(ind15m,1) = 1;
Xh(ind30m,2) = 1;
Xh(ind60m,3) = 1;
Xh(ind90m,4) = 1;
Xh(ind120m,5) = 1;
Xh(ind152m,1) = 1;
Xh(ind302m,2) = 1;
Xh(ind602m,3) = 1;
Xh(ind902m,4) = 1;
Xh(ind1202m,5) = 1;
X(ind15m,1) = 1;
X(ind30m,2) = 1;
X(ind60m,3) = 1;
X(ind90m,4) = 1;
X(ind120m,5) = 1;
X(ind152m,6) = 1;
X(ind302m,7) = 1;
X(ind602m,8) = 1;
X(ind902m,9) = 1;
X(ind1202m,10) = 1;
n = length(a(:,1));
out_data = zeros(n,12);
nsig = 0;

The dimensions of the X and Xh matrices in this case stay the same for all genes.

The rest of today was spent continuing to make changes to the MATLAB script for the deterministic model. Nick succeeded in separating the general parameter estimation function into four parts:

  • Input and labeling of the master sheet containing the network, log2 fold change concentrations for each strain, optimization parameters, production rates, degradation rates, network b's, and simulation times.
  • The call for the general least squares error and optimization
  • Generating graphs
  • Creating the output

The input of the mastersheet in this script is different from that in the previous general parameter estimation function. In the actual input sheet, the log2 concentrations were divided into several sheets (one for each deletion strain) and inputted sheet by sheet into MATLAB. In addition, the log2 concentrations for each flask for each timepoint were inputted rather than the average log2 concentrations for each timepoint. Indicators for the timepoints, strain name, and strain index were added to the optimization parameters sheet. This required modifying the for loop for inputting the optimization parameters.

The next steps with the deterministic model include making some changes to the general least squares error script where the error = ((log2(x1) - d1).^2) line of code needs to be modified to take into account the replicates in the log fold change data for each timepoint for each strain.

Katrina Sherbina 21:22, 23 May 2012 (EDT)

May 24, 2012

In order to modify the error calculation in the general least squares error function (in MATLAB) to take into account the change from using average log fold changes for each timpepoint to using all of the data, several modifications were made to other scripts. (To make any of these changes, I first dissected the various functions to understand the various parts more.)

The log fold change data had to be imported separately for each strain (this modification was made earlier in the week):

[log2FC(1).d,TX1]      = xlsread(input_file,'wt_log2FC');
[log2FC(2).d,TX1]      = xlsread(input_file,'dCIN5_log2FC');
[log2FC(3).d,TX1]      = xlsread(input_file,'dGLN3_log2FC');
[log2FC(4).d,TX1]      = xlsread(input_file,'dHMO1_log2FC');
[log2FC(5).d,TX1]      = xlsread(input_file,'dZAP1_log2FC');

Nevertheless, averages for each timepoint were later calculated for the purposes of later plotting standard deviations on the time versus log fold change plots.

The driver to detect active genes will be taken out. All of the genes in the network are considered active genes. The assignment of a number to each active gene was done with the detect active genes script. Now the active changes are "detected" in the matrices with the log fold change data:

active          = find(log2FC(1).d(2:end,1));

This array is called for in the general network dynamic function. The ode45 function in matlab approximates a solution to the differential equation defined in the general network dynamic function.

Previously, the error was calculated using the model and the average log fold changes for each timepoint from the data. However, this error calculation needs to be changed so that the unaveraged log fold changes can be used. A preliminary equation that was experiment with was

myerror = ((log2(x1) - d1([1 5 10],:)).^2)+((log2(x1) - d1([2 6 11],:)).^2)+
((log2(x1) - d1([3 7 12],:)).^2) +((log2(x1) - d1([4 8 13],:)).^2);

The problem here is that the equation does not take into account the last replicate for the t30 timepoint (because t30 has five replicates while the other timpeoints have four replicates).

Another concern is the actual data d and d1 that is being fed into the general least squares error function and other functions. For now, both d and d1 correspond to just the wildtype data. However, we will want to use the deletion strain data for the model as well.

Katrina Sherbina 21:29, 24 May 2012 (EDT)

May 25, 2012

The functions written in MATLAB for the deterministic model were modified to include all of the data (not just the average log fold changes for each timepoint) in the various calculations.

Modifications were made in the parameters script and the error calculation (now called errormat) in the general least quares error function. The following lines of code were added to the script:

% % The first row of the log2FC data indicating all of the replicate timepoints
    td               = (log2FC(1).d(1,:));
% % Finds the indices in td that correspond to each timepoint in tspan.
    tindex(1).indx   = find(td == tspan(1));
    tindex(2).indx   = find(td == tspan(2));
    tindex(3).indx   = find(td == tspan(3)); 
    n_times          = length(tspan);
% % Log2FC data for the wildtype
    d                = (log2FC(1).d(2:end,:))';
% % The average log2FC for each timepoint for each gene.
    means = zeros(length(log2FC(1).d(2:end,1)),n_times);
    for iT = 1:n_times
        means(:,iT) = mean(log2FC(1).d(2:end,tindex(iT).indx(1):tindex(iT).indx(end)),2);
    end
% % Standard deviations for each timepoint for each gene. Necessary for the graphs 
% % that will be produced.
    sigmas = zeros(length(log2FC(1).d(2:end,1)),n_times);
    for iT = 1:n_times
        sigmas(:,iT) = std(log2FC(1).d(2:end,tindex(iT).indx(1):tindex(iT).indx(end)),0,2);
    end

It is necessary to still find the standard deviations and the means to be able to produce graphs for each gene of the log fold change versus time with the upper and lower error bounds for each timepoint.

The error calculation in the general least squares error function was modified with the following lines of code:

errormat = 0;
for iT = 1:length(tspan)
    for iF =  1:length(tindex(iT).indx)
        errormat = errormat+(log2(x1(iT,:))-d(tindex(iT).indx(iF),:)).^2;
    end
end
% This is the sum of all the errors
L     = sum(errormat(:));

In the general least squares error function, a modification was also made to the first figure produced (with the counter and lse_out). At first, all of the data (not the average log fold changes) was being used to specify the second subplot in the figure which fits the model to the data. When viewing the figure, the model was being fit to only three of the thirteen sets of data points in the second subplot. There were thriteen sets of data points (in columns) since there are thirteen columns in the data due to the replicates in each timepoint. Instead of using all of the data, the code to produce the subplots was modified to instead use the average log fold change for each timepoint:

if(100*round(counter/100)==counter)
   figure(1),subplot(211),plot(theta,'d'), title(['counter = ' num2str(counter) '  lseout = '  num2str(lse_out)])
   subplot(212),plot(means','*'),hold on,plot(log2(x1)), hold off,pause(.1)
end 

The script specifying the plots for each gene of log fold change versus time was modified. The if else statement having to do with cssigflg was removed. This designation had to do with the concentration sigmas (standard deviations) inputted into MATLAB when they were still in the input excel spreadsheet. However, since the standard deviations are now being calculated in MATLAB, there is no need to determine the kind of the plot to be produced depending on the value of cssigflg. The following is the new code for the plots:

if igraph == 1
        concd = 2.^d(:,active);
        error_up = (means(active,:)+1.96*sigmas);
        error_dn = (means(active,:)-1.96*sigmas);
        for ii=1:n_active_genes
            figure(ii+2),hold on
            plot(time,log2(model(:,ii)),td,d(:,active(ii)),'o',tspan,error_up(ii,:),'r+',tspan,
             error_dn(ii,:),'r+','LineWidth',3),axis([tmin tmax -3 3])
            title(TX1{1+(ii),2},'FontSize',16)
            xlabel('Time (minutes)','FontSize',16)
            ylabel('Expression (log2 fold change)','FontSize',16)
        end
end

The global statement at the top of each script or function was also changed due to the aforementioned changes:

global A prorate degrate active inactive tspan d wts b alpha lse_out penalty_out counter parms parmnames tindex means

Further work will need ot be done on the plots. In the plots for MAL33 and ZAP1, the upper error bounds are not shown. The y axis may not to be adjusted to be larger than [-3,3]. Also, the model for HAP5, the model goes beyond the error bounds for the t60 timepoint.

In the output excel spreadsheet, there are no log2 optimized concentrations nor network optimized B's for FHL1, SKO1, or SWI6.

In addition to dealing with the aforementioned concerns, we need to explore how to include the deletion strain data in the model.

Katrina Sherbina 20:23, 25 May 2012 (EDT)

Week 3

May 29, 2012

Copies were made of all of the functions and scripts in order to keep the original scripts that worked last week when making modifications to these scripts today. The ending _strains was added to each m file.

A line of code was added to the estimation driver that indicates the sheet with the data of the deletion strain for which the script is being run at a given time. Since the model is to be found for each one of the strains, it makes it easier in the long run just to import the data for the deletion strain that the code will be run for.

This change was made in the process of spending the day changing the general network dynamics function to be able to run the model for each of the deletion strains. It was necessary to delete the information in the weights matrix and the degradation rates array that corresponded to the gene that was deleted in the deletion strain of interest. One of the lines of code added, finds the index of the gene deleted in a given strain by matching it to the sheet number in the input corresponding to the log fold change data for the deletion strain. To be able to incorporate this line, the variables num and indices were added to the global command line.

When running the entire code, the first error come up was a mismatch of dimesnions in the line WXB = -W*zz+B which computes the exponent in part of the differential equation that is a sigmoid function. This error was due to the fact that B (which corresponds to b) had 18 rows while the other two arrays had 21. This occurred because b was being recomputed in the general least squares error function. As a result, the recomputed b was renamed to bb.

However, another error occurred at line 43 in the LSE_strains script. The matrix dimensions do not agree. It's uncertain why these errors are occuring because after comparing each line of code Nick and I seem to be using the same scripts.

More troubleshooting will be done tomorrow to address the newest error.

Katrina Sherbina 20:57, 29 May 2012 (EDT)

May 30. 2012

The errors that were encountered yesterday were fixed with some changes to the general network dynamic function and the calls to various functions. The reason why there was a mismatch in dimensions in the line WXB = -W*zz+B in the network dynamic function is that B was the same array as b. Intially, b was a 21x1 vector of 0's. However, in the general least squares error function b changed to a 18x1 vector of 0's because there 18 of the 21 total number of genes in the network regulate the expression of other genes. However, B needs to be a 21x1 vector of 0's to be able to compute WXB. For this to be possible, a line of code was added to the general network dynamics function instead of renaming the recalculated b in the general least squares error function:

B = zeros(n_genes,1);

As a result, n_genes was added to the global line.

The other error that occurred was solved by changing the function calls. There are currently two sets of scripts (one set is the original set that was composed last week and the other is the set for which the sheet number of the strain being worked on is specified in order to import the correct log fold change data). However, within the second aforementioned set of scripts, the LSE script and a few other functions called for functions from the first set of scripts. All function calls were changed to those corresponding to the scripts in the second set of script previously discussed.

However, with these changes, another problem arose. After running the fmincon function in the LSE script, the numbers composing the w1 array did not change as expected. Some of the numbers differed only in the ninth or so decimal place. This resulted in many of the genes having the same network optimized weights. This was not the case when the scripts from last week were executed.

To explore this problem, I started troubleshooting from the set of scripts from last week making changes that lead to the newest set of scripts one by one. After doing so, it seems that the problem that was observed arose when the general dynamic function is changed from what it was originally to the one constructed yesterday. This problem was evident in the first subplot of figure 1 plotting all of the theta values. Most of the 71 theta's stayed in practially the same line centered at 0. Hoever, what is odd is that the new general dynamic function produces the same dz vector as the old network dynamic function. In addition, the same x is found after applying the ode45 function to the new general network dynamic function as when applying the ode45 function to the original general network dynamic function. Despite this, the w1 vector (which is being recalculated by fmincon from the original vector corresponding to w0) is not being recalculated correctly by the fmincon function. This problem will be troubleshooted further tomorrow.

Towards the end of the day, the MATLAB scripts to do the ANOVA and p value corrections for the all strain and two strain comparisons were revisted. The ability to pause at a plot and to save a plot as a .jpg was added to the lines of code to produce the plots of the data and model for each gene having a p value of less than 0.05:

 pause on
   if pval<0.05
       figure(1),plot(t,Y,'bo'),hold on,plot(t,Ym,'r+','LineWidth',3),plot(t,Yh,'k^','LineWidth',3),
           title([b{ii+1,1} '   W = ',num2str(W),' pval = ',num2str(pval)]),...
           xlabel('time, minutes'),ylabel('log fold change'),legend('data','model'),drawnow
       hold off
       nsig = nsig + 1;
       [ii,nsig]
       drawnow
       %Allows you to pause at a graph for as long as you do not press a key on the keyboard.
       pause
       %Save as a .jpg
       saveas(figure(1),[b{ii+1,1} '.jpg'])
       %%Save as a .fig
       %saveas(figure(1),[b{ii+1,1} '.fig'])
       %%Captures the figure being currently displayed to put into the movie.
       %movieArray(nsig) = getframe(gcf);
   end 

Katrina Sherbina 21:22, 30 May 2012 (EDT)

May 31 2012

The errors arising with w1 vector were were troubleshooted. First, the code including the new general network dynamics function was run through the fmincon function for 5 different networks: an identity matrix, a matrix with two diagonals of 1, a matrix with a diagonal of 1 and alternating 1's and 0's above the diagonal, a matrix with a diagonal of 1's and the first row of 1's, and a matrix with a diagonal of 1's and the first column of 1's. The results were as follow.

-The output (w1 vector) produced with the network matrix with a diagonal of 1's and the first row of 1's had the most number of entries that were not identical (the first 21).

-With the exception of the network matrix just mentioned, the number of unique entries within the w1 vector for the most part increased as the number of edges in the network increased:

  • identity matrix: 21 edges, 1 unique entry in w1 vector
  • matrix with two diagonals of 1: 35 edges, 2 unique entries in w1 vector
  • matrix with a diagonal of 1's and the first column of 1's: 41 edges, 2 unique entries in w1 vector
  • matrix with a diagonal of 1 and alternating 1's and 0's above the diagonal: 121 edges, 11 unique entries

-Both the network matrix with a diagonal of 1's and the first row of 1's and the network matrix with a diagonal of 1's and the first column of 1's had 41 edges. However, there were 21 unique entries for the former network and only 11 unique entries for the later network in the w1 vector.

Then, I ran each of the matrices through the code but setting the thresholds to 1.00E-10 because one of the messages that popped up in the command window was that the optimizaiton was terminated because "the magnitude of directional derivative in search direction less than 2*options." I obtained pretty much the same results. Between some corresponding entries in the corresponding w1 vectors, there may have been a difference in the fifth decimal place.

I then ran each of these five networks using the original general network dynamics function. All of the entires in the w1 vectors for each of the networks were unique. At which point, the general network dynamics function was then revisted because that is most likely whyere the problem lay.

It was observed that the parms_used variable changes within the for loop in the original network dyanmics code but it stays at 0 in the new general network dynamics code. Therefore, the line parms_used = parms_used + nAii; was added inside the for loop to calculate W in the new general network dynamics function. In the output, the network optimized weights for each gene were different (they were the same for a majority of the genes when the w1 vector was not being properly computed as described before). However, the new w1 vector obtained did not match the one calculated using the old general network dynamic function. Then the output of the general network dynamics function was looked at more closely. It was observed that the computed dz was different with in the new general network dynamics code (with parms_used not fixed) in comparison to the dz computed by the original general network dynamics code. This will need to be looked into.

In addition, the plots generated in the two strain comparison and all strain comparison code will need to be changed to fit a polynomial through the data for each strain being compared and to include the standard name as well as the systematic name for each gene for which a plot is being produced.


Katrina Sherbina 21:40, 31 May 2012 (EDT)

June 1, 2012

Yesterday, using two different functions to specify the general network dynamics were produced different results (different w1 vectors, different b's, and different network optimized weights). Looking at yesterday's output again today, it was observed that the w1 vector had 21 0's at the end. Also, all of the network b's outputted were 0. This indicates that the b's were not changing despite the intial parameters indicating that b is not fixed. The problem lay in the general network dynamics function. To stop the b's from being fixed, the following line was added before the for loop in the function:

B(i_forced) = b;

To check whether or not the two different general network dynamics function were producing the same results, the two outputs after solving the differential equation with ode45 for the original general network dynamics function and the new general network dynamics function were plotted on the same graph (x versus t). The outputs of both functions matched.

The entire new code for the deterministic model was rerun. This time a majority of the b's in the output were nonzero with the exception of three for genes that did not regulate other genes. This result was expected.

The original code for the deterministic model was rerun to compare with the output of the new code. For the most part, the b's and the optimized weights in the output were similar in magnitude and the same in signs to those in the output of the new code.

The rest of the day was spent trying to figure out how to run the model for each of the sets of deletion strain data. Rather than creating multiple for loops within each of the scripts and functions, one for loop was experimented with in the estimation driver that would run all of the scripts and functions for each of the deletion strain data sets. The first for loop tried with the input was

input_file_name = 'New_Input';
S = ['wt   '; 'dcin5'; 'dgln3'; 'dhmo1'; 'dzap1'];
strain = cellstr(S);
input_file  = [input_file_name '.xls'];
[type, sheets] = xlsfinfo(input_file);
Common_Parameters;
for ii = 1:length(sheets)
   for jj = 1:length(strain)
       if strcmp(sheets(ii),strain(jj))
          sheet = strain(jj);
          num = ii;
          Strain_Parameters;
          LSE_strains;
          Graphs_strains;
          Output_strains;
       end
   end
   continue
end 

The original Parameters script was split into two:

  • The Common_Parameters script imports all parameters from the input sheet that are the same for all strains.
  • The Strain_Parameters script imports the parts of the input specific to the strain (like the log fold changes) and makes computations that are specific to the strain.

The Common_Parameters script was created to keep the code more efficient by not reinputting parts of the input sheet that are the same for all strains each time the for loop is executed.

This for loop would run through the whole deterministic model code for the widltype producing all of the specified graphs and the output sheet. However, instead of moving on to the next (deletion) strain data set, the loop was terminated and an error appeared for the line strcmp(sheets(ii),strain(jj)) indicating that the matrix dimensions did not match.

The for loop was then modified so that it created an array of sheet numbers corresponding to the sheet number of each of the strains indicated and an array of sheet names corresponding to the sheet name that contained the data for each of the strains. Then another for loop was created that ran all of the scripts and functions for each pair of sheet numbers and sheet names:

input_file_name = 'New_Input';
S = ['wt   '; 'dcin5'; 'dgln3'; 'dhmo1'; 'dzap1'];
strain = cellstr(S);
input_file  = [input_file_name '.xls'];
[type, sheets] = xlsfinfo(input_file);
Common_Parameters;
sheet_name = cell(length(strain),1);
sheet_number = zeros(length(strain),1);
for ii = 1:length(sheets)
   for jj = 1:length(strain)
       if strcmp(sheets(ii),strain(jj))
          sheet_name(jj,1) = strain(jj);
          sheet_number(jj,1) = ii;
       end
   end
end 
sheet_name = cellstr(sheet_name);
for ii = 1:length(sheet_name)
   sheet = char(sheet_name(ii,1))
   num = sheet_number(ii,1)
   Strain_Parameters;
   LSE_strains;
   Graphs_strains;
   Output_strains;
end

When running this script, the model was computed for each of the strain data sets. The output for the wildtype with this script was the same compared to the output for the wildtype that was computed earlier in the day with the code that just ran for one strain. The network optimized weights and the network b's were the same in both outputs.

The outputs for the deletion strain data differed from that of the wildtype as expected. The network b was 0 for the gene which was deleted to obtain a given output.

The graphs that were produced of log fold change versus time plotted the model for each of the strain data sets on the same plot for each gene. A next step would be to also separate these graphs so that a separate set of plots for each gene is produced for each strain data set.

In addition, the graphs from the statistical tests need to be revisted to fit a line through the data points. Rather than fitting a polynomial using Dr. Fitzpatrick's code, the symbol for the plot of the data set will be removed in the code so that a continuous line is produced through the data points.

Katrina Sherbina 21:22, 1 June 2012 (EDT)

Week 4

June 4, 2012

Today two problems became apparent with the code that I had left off on last Friday (June 1st):

  • While the the b's corresponding to the deletion strains were being zeroed out, the rows and columns corresponding to the deletion strains in the weights matrix were not being zeroed out. There were non zero values for these genes supposedly deleted in the model in the network optimized weights.
  • The process to go through the model with the deletion strain data treated the data for each strain separately when in revamping the code we should be using all of the data for all of the strains simulatneously.

To start fixing the second of the two problems, the second of the two for loops in the estimation driver was modified taken out of the driver and put into the general least squares error function. The data without the log fold changes (d) and the errormat calculation were put into this loop. Also, the call for the ode45 function was also put into this loop. In doing so, the LSE was being computed as a sum of the difference between the model and data squared over flask, times, genes, and strains whether than just over flasks, times, and genes as before.

Know I'm a little bit confused about the how the output should look. In making this change, it seems that the rows and columns corresponding to the genes deleted in each of the deletion strains are being zeroed out for all of the deletion strains at once. Therefore, I'm not sure how to have a separate output of the results of running through the model for each deletion strain.

Katrina Sherbina 20:39, 4 June 2012 (EDT)

June 5, 2012

We spent the majority of the day in Dr. Dahlquist's lab scanning the microarrays that were prepared from the mRNA degradation experiment with GenePix Pro. We used the normalization procedure that was developed last summer to normalize all of the chips using both within array and between array normalization. See the protocal in the following link: Dahlquist:Microarray Data Processing in R. Nick and I both normalized the microarray data separately to make sure that we obtained in the same results. Both of our final outputs are the between array normalization were the same.

Katrina Sherbina 20:22, 5 June 2012 (EDT)

June 6, 2012

Dr. Fitzpatrick asked me to work on a problem aside from the deterministic model involving the data from the 72-hr growth experiment performed a couple of weeks looking at the growth rate of S. cerevisiae wildtype and S. paradoxus in both 13 degrees C and 30 degrees Celsius. When Dr. Dahlquist crunched the numbers, she found that S. paradoxus grew better than S. cerevisiae in both temperatures. My task today was to use a linear model to determine whether or not the difference in the growth rates between S. cerevisiae and S. paradoxus are significantly different. The result of this analysis performed in SPSS using the linear regression tools was that the growth rate (the slope of the data consisting of a fixed component, a temperature dependent component, and a strain dependent component) was siginficant. The next step in analyzing the data is to determine whether or not it is significant that S. paradoxus grew faster in the cold that what would be expected considering its fast growth rate in the warm? I will be working on constructing a matrix for the linear model for this question tomorrow.

The other tasks of today included working more on the deterministic model. The code from Monday was redudant in that the tindices and d's (the log fold change data) were computed twice in both a for loop in the Parameters_all_strain_data code and the general_least_squares_error_sum_over_strains function. To remove this redudancy, the for loop in the Parameters script was redone as follows

for ii = 1:length(sheet_number)
   sheet = sheet_name(ii,1);
   num = sheet_number(ii,1);
   td = (log2FC(ii).d(1,:));
   tindex(1).t(ii).indx   = find(td == tspan(1));
   tindex(2).t(ii).indx   = find(td == tspan(2));
   tindex(3).t(ii).indx   = find(td == tspan(3));
   d = log2FC(ii).d(2:end,:);
   data(ii).d = d;
   for iT = 1:n_times
       means(ii).m(:,iT) = mean(data(ii).d(:,tindex(iT).t(ii).indx(1):tindex(iT).t(ii).indx(end)),2);
       sigmas(ii).s(:,iT) = std(data(ii).d(:,tindex(iT).t(ii).indx(1):tindex(iT).t(ii).indx(end)),0,2);
   end
end

Then tindex and data was added to the global statement. The tindex and d computations were then removed from the for loop in the general least squares error code.

The code were it stands right now uses all of the data for all strains in finding the model. The issue has been trying to write separate outputs and graphs depicting the model for each strain. Dr. Fizpatrick suggested making a for loop in the LSE line of code to output the model for each strain. This loop currently includes the model outputted when using the ode45 function to solve the differential equation defined by the generla network dynamics, the code to make the log FC graphs versus time for each gene, and calls to other scripts to make the output. Both the graphs and the output were crated after some tweaks to make sure that vectors and matrices were the right dimensions. However, each of the outputs produced (one for each strain) were exactly the same in the network b's and optimized weights. In fact, they all looked at the output for just the wildtype. In addition to working on the linear model described earlier, tomorrow will be spent also trying to correct the outputs. We will check to make sure that the problem is in the way the output is being written and not in how the model is being computed for each strain.

Katrina Sherbina 21:34, 6 June 2012 (EDT)

June 7, 2012

Part of the morning was spent testing a second hypothesis regarding the 72 hour growth experiment using a linear model. Using SPSS, I tested whether or not the change in the growth rate between 13 degrees Celsius and 30 degrees Celsius conditions for S. paradoxus was significantly different from the change in the growth rate between these temperatures for S. cerevisiae. This involved modifying the matrix created yesterday to include 8 parameters (4 intercepts, 2 slopes for cold conditions, and 2 changes in slope between the cold growth rate and hot growth rate). Both a full model and a hypothesized model was computed in SPSS where the hypothesized model had one less parameter (one less change in growth rate) than the full model. The full model adn hypothesized model residual sum of squares was used to calculate and F statistic and p value. The result was that the change in growth rate for S. paradoxus as a result of shifting from hot to cold is significantly different from that of S. cerevisiae. SPSS was not able to compute the p value because it was so small. An external p value calculator was used but it could only compute that the p value was less than 0.0001.

I also went back to the tast of outputting graphs of the log fold changes versus time for each gene for different strain data sets. I tried Dr. Fitzpatrick's suggestion to get rid of the symbol in the designating the data poitns in plot line of code in the wildtype deletion strain compare and two deletion strain compare scripts. However, this produced bizarre graphs that made it impossible to tell what the data values were. Therefore, I went back to the polynomial fit script that Dr. Fitzpatrick used in the past and I modified it to display a polynomial fit for two strains (wildtype and deletion strain or deletion strain and deletion strain). Dr. Dahlquist wanted the title of the graph to include the standard name of the gene. However, at this point, the title contained the systematic and standard names of the gene, the f stats for the gene for both sets of strain data, and the p values for the gene for both sets of strain data. The title could not fit onto one line and threw off the dimensiosn of the plot. Tomorrow, I will look into breaking the title into two lines or maybe having a subtitle with the statistics to keep the systematic and standard name in the same line as the title.

I did not put much work inot the deterministic model today. Tomorrow, I will be getting the code that Nick finished that made it possible to get different outputs showing the different model for each strain. We will probably end up cleaning up the parameters script to take advantage of the strcmp command to import optimization parameters among other small changes.

Katrina Sherbina 21:40, 7 June 2012 (EDT)

June 8, 2012

I spent a part of the morning working on the polynomial fit code so that the title would just include the systematic and standard names of the genes while the caption beneath the x axis label would have the F statistic and p value for each strain being analyzed. While I was able to format the title and the caption, the polynomial fit code was put aside and the original two deletion strain compare and wildtype deletion strain compare script was revisted. Both codes were changed similarily to output graphs for each gene with a significant change in expression that plotted the data points, continuous lines through models of both strains, and the hypothesized model. In order to do so, the times needed to be separated to designate which corresponded to which strain. This was added inside the loop in the wildtype deletion strain comparison and outside the two deletion strain comparison:

   ts1 = [ones(n15,1)*15;ones(n30,1)*30;ones(n60,1)*60;ones(n90,1)*90;ones(n120,1)*120];
   ts2 = [ones(n152,1)*15;ones(n302,1)*30;ones(n602,1)*60;ones(n902,1)*90;ones(n1202,1)*120];
   t   = [ts1;ts2];
   [tsort,is] = sort(t);

Inside the loop for both sets of code, the way Y was calculated was separated for each strain:

   Ys1 = a(ii,indx)';
   Ys2 = a(ii,indx2)';
   Y   = [Ys1;Ys2];

Also, calculation for Ym (the full model) was split into two: one for each strain compared.

   Ym1 = X(ind15(1):ind120(end),1:5)*beta(1:5,1);
   Ym2 = X(ind152m(1):ind1202m(end),6:10)*beta(6:10,1);

The plot command was then changed:

pause on
   if pval<0.05
       figure(1),plot(ts1,Ys1,'ro'),hold on,plot(ts2,Ys2,'mo'),plot(ts1,Ym1,'r','LineWidth',2),... 
           plot(ts2,Ym2,'m','LineWidth',2),plot(tsort,Yh(is),'k','LineWidth',2),title([b{ii+1,1} '/' b{ii+1,2},...
           '   W = ',num2str(W),' pval = ',num2str(pval)]),xlabel('time, minutes'),ylabel('log fold change'),...
           legend([strain1 ' data'],[strain2 ' data'],[strain1 ' model'],[strain2 ' model'],'hypothesized model'),drawnow
       hold off
       nsig = nsig + 1;
       [ii,nsig]
       drawnow
       %         Save as a .jpg
       saveas(figure(1),[b{ii+1,1} '.jpg'])
   end

Katrina Sherbina 19:18, 8 June 2012 (EDT)

Week 5

June 11, 2012

Today, we began working on the two abstracts we will be submitting to the conference held by the Society for Mathematical Biology. Right now, I have a first draft of the abstract for the poster that will deal with the work we have done with the deterministic model.

The other part of today was spent continuing to try to debug the code for the deterministic model. The outputted weights of the current code for the model seem to be matching the outputted weights for when just the wildtype data was used as was seen in the graph Nick created plotting the weights against a numerical index for the current weights output and the output for each of the deletion strains in which the data for one strain was being used at a time. While we do want one set of network weights and network b's for all of the strains, the weights should not be identical to those for the previous wildtype only model. We're not sure exactly where the problem is in our code. We've already determined that the rror calculation in the general least squares function that fmincon calls upon is being updated for each strain data set, for each timepoint, for each replicate. We will be troubleshooting this problem further tomorrow.

In addition, Chiddy today emailed me two sets of microarray data for the mRNA decay experiment that differ in the way the PMT was calculated. The goal is to be able to normalize this data and compare it to the normalized microarray data we obtained last week to see which way is better to calculate the PMT when scanning the microarrays. The problem is that our current normalization assumes that the expression of most of the genes remains unchanged. However, in this mRNA decay experiment, the end result is that all of the mRNA has decayed so that the spots on the microarrays should be getting progressively more intensely green. We will need to talk about how to properly normalize the data so that we can determine whether or not to perform replicates of the experiment to be able to later determine mRNA decay rates for the transcripts of the genes being analyzed.

Katrina Sherbina

June 12, 2012

I focused today mainly on analyzing the most recent microarray data from the first mRNA decay experiment that Dr. Dahlquist performed. There were three sets of data differing in how the PMT was set: manually for each arrray, automatically by the program for each array, or one PMT for each array based on an average. We decided not to hold off on the within array normalization and between array normalization (the protocol that was created last summer) because these kind of normalization procedures are based on the assumption that a majority of the genes will not have a significant change in expression. However, we do expect to see a change in the mRNA decay experiment.

I began by first making MA plots for the non-normalized data (one plot for each GPR file). I used a script similar to the one that was used last summer where the M values and A values were calculated by hand from the RG file that was created when reading in the targets. I did have to change the M and A matrices (after the tapply function was performed) to data frames so that I could deleted the controls Arabidopsis and 3XSSC. To deleted the controls, I first specified the the rownames (gene names) within R rather an importing a sv file of the gene names as was done previously:

RGt<-tapply(RG$R[,1],as.factor(GeneList),mean)
geneID <-row.names(RGt)
MG2<-as.data.frame.matrix(MG)
#lfc is the matrix of log fold changes before the tapply function has been done.
colnames(MG2)<-colnames(lfc)
rownames(MG2)<-geneID
#The same thing was done to make AG (intensities) a data frame.

Then I used subsetting to get rid of the controls:

MG3<-subset(MG2,row.names(MG2)!="Arabidopsis")
MG4<-subset(MG3,row.names(MG3)!="3XSSC")
#The same thing was done to get rid of the controls in AG.

In plotting the A values versus the M values I noticed that the plots liked like what I would expect them to look after within array normalization. A majority of the plots seemed centered at 0.

I also did box plots of the M values for each timepoint for each of the data sets. Again, I saw that a majority of the box plots were almost centered at 0.

Then, as Dr. Fitzpatrick advised, I did a linear regression of the mean M (lnG-lnR) for each timepoint for each data set versus time. I tried to do this at first in R. First, I recalculated the M values by finding the log2((RG$G-RG$Gb)/(RG$R-RG$Rb) for each of the data points before performing the tapply function. Since some of the values were NaN, Inf, or -Inf, I ran into a few snags using colMeans. The designation na.rm=T did not seem to work because there were Inf and -Inf values. I tried to change these values to NaN within R but could not get a for loop consisting of an if statement to work. So I exported the data and made the change in excel. I then reimported the data. I could successfully get the equation for perofrming the linear regression on meanM versus time for each data set. However, I had difficulty trying to plot the actual data points and the regression. Finally, I just made plots with trendlines of meanM versus time for each data set in Excel. It was hard to see any trend in the mean M values over time. The slopes for two plots (automatic PMT and average PMT) seemed close to 0. However, it is difficult to infer anything from these plots sine the R^2 value for each regression was week.

Tomorrow, I will try to make plots of the M values (lnG-lnR) of each gene versus time for each data set.

Katrina Sherbina 20:59, 12 June 2012 (EDT)

June 13, 2012

Todya, I continued to analyze the mRNA decay experiment microarray data. We decided to do plots of the M values (lnG-lnR) versus time for each gene. However, observing any kind of trend or biase among the arrays this way was already considered a stretch from the onset but we decided to try anyway. I made the plots specifically for the average PMT data. First, I exported the M values calculated in R and loaded them into MATLAB as a .csv file. Within MATLAB, I constructed a loop that plotted the M values (from the average PMT data) versus time (timepoints in the experiment) for each gene and also a line of best fit through the data points for each gene using the polyfit function. As expected, there was no observable bias across all of the genes. One of the problems may have been that there were no replicates so the lines of best fit probably did not fit the data points well. After sharing these graphs with Fitzpatrick, there is not much else to interpret from this data. We need to talk with Dr. Dahlquist about the experiment and the data more at the next lab meeting.

The rest of today was spent continuing to work on the abstract for the mathematical biology conference in July that is due this Friday.

Katrina Sherbina 20:47, 13 June 2012 (EDT)

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