- display the pseudoarray image
for the current HP sample
- display various scatter plots
of selected pairs of HP samples
- display both ratio and
intensity histograms of HP sample data
- display expression profile
plots of genes or gene subsets
You may switch between different representations of the microarray spot pseudoarray image. It may be viewed as several different types of pseudo images including an intensity gray value and a pseudo-color Red/Black/Green image for ratio (HP-X/HP-Y) and Zscore (HP-X - HP-Y) data. The p-Value results of comparing a HP-X 'set' with a HP-Y 'set' of samples, or the CV of the HP-EP 'list can be displayed as a color spectrum pseudoarray image.
Depending on the origin of the array data, it may have the same verisimilitude as the original arrays. Otherwise, it is displayed in a generic pseudoarray image containing grids that will fit the window - these are not the same as the original array image (see . However, the pseudoarrays are useful to getting a rough idea of the global changes in the data between arrays and how may genes pass the data filter.
When enabled using one of the commands in the Section 2.4.5 Clustering menu, cluster data appears as blue circles or squares drawn as overlays on the pseudoarray image. These options are discussed in the section on clustering. If you are doing clustering K-means clustering, the current cluster is displayed in the scatter plot if the latter is active.
Scatter plots, ratio and intensity histograms of the mean (HP-X/HP-Y) or (HP-X/HP-Y) 'set' data, or the F1/F2 or Cy3/Cy5 data. F1/F2 or Cy3/Cy5 plots are available if the data exists in your particular database. That might be the case with replicate spots or with Cy3/Cy5 data. If the normalization is set to a Zscore or log mean mode, it will compute Zscore scatter plots and histograms.
Clicking on spots in an array image or points in scatter plots sets the current gene and will bring up data on the gene or (optionally) access corresponding data from GenBank, UniGene, mAdb Clone, etc. databases in a popup Web browser. Clicking on a bin in a ratio or intensity histogram plot filters out all genes except for those in the range of that bin.
Expression profiles plots of selected genes or subsets of genes for all samples in the HP-E list. These are active plots with data reported when the user clicks in the plot.
Clicking on a spot (i.e. gene) in the microarray pseudo image or on a point (i.e. gene) in the scatter plot, it will define that gene as the "current gene" that is used in other operations. The current gene is indicated in both plots with a green circle around it. Similarly, you may modify the 'Edited Gene List' from either the pseudoarray image or the scatter plot. When viewing is enabled, it overlays those genes with magenta squares.
Figure 2.4.4 Plot menu - selecting Ratio Pseudoarray image. This displays a pseudocolor show in the scale on the left that indicates the ratio of the value of the HP-X sample / HP-Y sample (or 'sets' if the option to use HP-X and HP-Y 'sets' is enabled.) If The data is Cy3/Cy5 data, then this displays the ratio of the ratios using the current normalization. Various other pseudoarray image representations could be used.
Pseudograyscale intensity [RB] - display a pseudograyscale
microarray pseudoarray image for the current H.P. where higher
intensity spots are mapped to black.
Pseudocolor Red(X)-Yellow-Green(Y) HP-XY ratio or Zdiff [RB] -
display the HP-X and HP-Y microarrays as a pseudocolor image as
either a ratio (HP-X/HP-Y) or Zdiff (HP-X - HP-Y) if Zscore
normalization is in effect. The HP-X (red) and HP-Y (green)
components are added together so that high total
intensity values are yellow and low values are black. High
HP-X/HP-Y ratio values are more red and low values are more
green.
Pseudocolor Red(Cy5)-Yellow-Green(Cy3) Cy3/Cy5 data [RB] -
display the Cy3 and Cy5 microarray channels as a pseudocolor
array image. The the Cy5 (red) and Cy3 (green) components are
added together so that high total intensity values are
yellow and low values are black. High Cy3/Cy5 ratio values are
more green and low values are more red. Note: this is only
available with Cy3, Cy5 ratio data. Note: if the "Use ratio
median corrections" is enabled, the Cy3 channel is scaled using
the to the domain of the Cy5 channel by (median Cy5/median Cy3).
Pseudocolor HP-X/Y ratio or Zdiff [RB] - display the HP-X and
HP-Y data as a pseudocolor array image as either a ratio
(HP-X/HP-Y) or Zdiff (HP-X - HP-Y) if Zscore normalization is in
effect. The high ratios or Zscores are red and low values are
green. Values in the middle are black.
Pseudocolor (HP-X,HP-Y) 'sets' p-value [RB] - displays each
spot as a pseudocolor proportional to the p-Value in a t-Test of
the HP-X 'set' vs HP-Y 'set'. This can only be used when the
"Use HP 'sets' ..." option is enabled and there are at least 2
samples in both the HP-X 'set' and the HP-Y 'set'. Note that
unless proper normalization and filtering is used to remove poor
quality data, some of the p-Values will not be significant and
there will be a high false-positive rate. Use this display with
that in mind.
Pseudocolor HP-EP 'list' CV (Coefficient of Variation)
[RB] - displays each spot as a pseudocolor proportional to
the CV of the spots within the subset of samples in the current
HP-EP 'list'. It is useful to look at the variation in a set of
replicate samples. Use the (Samples | Choose HP-X, HP-Y and HP-E
samples) command to define the HP-EP list of samples you want to
investigate.
Pseudocolor Cy3/Cy5 (or F1/F2) ratio or Zdiff [RB] - display
the Cy3 and Cy5 (or F1 and F2 for duplicate spotted grids)
corresponding spots for the same genes as a pseudocolor array
image as either a ratio (Cy3/Cy5) or Zdiff (Cy3-Cy5) if Zscore
normalization is in effect. The high ratios or Zscores are red
and low values are green. Values in the middle are black.
Flicker HP-X & HP-Y Pseudoimages [RB] - toggle flickering the
HP-X and HP-Y pseudograyscale intensity array images.
Use dual HP-X & HP-Y Pseudoimage [CB] - If have replicate
spots (F1,F2), toggle the display between F1 data in left grids
and F2 data in the right grids to mean HP-X data in the left
grids and HP-Y in the right grids for side by side comparisons.
"Scale pseudoarray image by
1/100 to zoom low-range values [CB] - rescales intensity and
(Cy3+Cy5) and (HP-X + HY-Y) (Red-Yellow-Green) plots to so
mid values are easier to visualize.
Table 2.4.4.1. Pseudocolors assigned to spots to represent data in the X/Y ratios or X-Y Zdiffs pseudocolor array images. Each color represents the normalized X/Y ratio or X-Y Zdiff depending on Normalization mode. The 9 colors of the boxes represent the normalized expression ranges.
| . | . | . | . | . | . | . | . | . | Normalization mode - RBG |
| bright green | . | . | dark green | Black | dark red | . | . | bright red | |
| . | . | . | . | . | . | . | . | . | Normalization mode - dichromasy |
| bright blue | . | . | dark blue | Black | dark orange | . | . | bright orange | |
| <0.250X | 0.307X | 0.400X | 0.571X | 1.000X | 1.75X | 2.50X | 3.25X | >4.00X | Ratio data |
| <-3.0 | -2.25 | -1.50 | -0.75 | 0.00 | 0.75 | 1.50 | 2.75 | >3.0 | Zscore data |
| <-0.99 | -0.742 | -0.495 | -0.247 | 0.000 | 0.247 | 0.495 | 0.742 | >0.99 | Zscore Log data |
Clicking on a particular gene will report its specific quantification and identification values (See Section 3.3 on gene quantification). If the Enable display current gene in popup genomic DB Web Browser option is set in the View menu, then it will also pop up a Web browser with the corresponding to the particular genomic DB data for that database if it exists.
The same data is shown in a variety of normalization and display formats.
Figure 2.4.4.1.1.1 Pseudoarray intensity image of median normalized intensities of the current HP sample (C57B6 virgin 10 weeks from MGAP database). The graylevel scale on the left edge of the pseudoarray image indicates the spot intensity. All pseudoarray images have scales that vary depending on the type of pseudoarray being displayed.
Figure 2.4.4.1.1.2 Pseudoarray intensity image of Zscore normalized intensities of the current HP (C57B6 virgin 10 weeks from MGAP database).
Figure 2.4.4.1.1.3 Pseudoarray intensity image of ZscoreLog normalized intensities of the current HP (C57B6 virgin 10 weeks from MGAP database).
Figure 2.4.4.1.1.4 Pseudoarray intensity image of ZscoreLog normalized intensities of the dual HP-X and HY-Y individual samples. The Plot menu Show Microarray submenu toggle "Use dual HP-X & HP-Y samples" option is set. HP-X is a C57B6 pregnancy day 13 and HP-Y is a Stat5a (-,-) pregnancy day 13.
Figure 2.4.4.1.1.5 Pseudoarray intensity image of ZscoreLog normalized intensities of the dual HP-X and HY-Y sample 'sets'. The Plot menu Show Microarray submenu toggle "Use dual HP-X & HP-Y samples" option is set. The "Use HP-X & HP-Y 'sets' option in the Samples menu. HP-X is the mean of three 'C57B6 pregnancy day 13' and HP-Y is the mean of three 'Stat5a (-,-) pregnancy day 13'.
Figure 2.4.4.1.2.1 Pseudocolor array image of median normalized X/Y ratios. HP-X is C57B6 pregnancy day 13 and HP-Y is Stat5a (-,-) pregnancy day 13. Each spot's color represents the normalized X/Y ratio depending on Normalization mode. The color of the box is one of 9 colors representing the normalized expression ranges and assigned according to the table "Ratio mode".
Figure 2.4.4.1.2.2 Pseudoarray color image of normalized X/Y 'set' mean value ratios. Mean of three HP-X C57B5 pregnancy day 13 samples and mean of three HP-Y Stat5a (-,-) pregnancy day 13 samples. Each spot's color represents the normalized X/Y 'set' ratios depending on Normalization mode. The color of the box is one of 9 colors representing the normalized expression ranges and assigned according to the table "Ratio mode".
Figure 2.4.4.1.2.3 Pseudoarray color image of X-Y Zdiffs. HP-X is C57B6 pregnancy day 13 and HP-Y is Stat5a (-,-) pregnancy day 13. Each spot's color represents the normalized X-Y Zdiff depending using the Zdiff normalization mode. The color of the box is one of 9 colors representing the normalized expression ranges and assigned according to the table "Zdiff mode".
Figure 2.4.4.1.2.4 Pseudoarray color image of X-Y Zdiff of log data. HP-X C57B5 pregnancy day 13 sample and HP-Y Stat5a (-,-) pregnancy day 13 sample. Each spot's color represents the normalized X/Y ratio depending on ZdiffLog with StdDev normalization mode. The color of the box is one of 9 colors representing the normalized expression ranges and assigned according to the table "ZdiffLog mode".
Figure 2.4.4.1.2.5 Pseudoarray image showing color-coded p-values for t-test comparison of HP-X and HP-Y 'set' samples. The HP-X and HP-Y sets both have 2 samples each (more is obviously much better). The data was normalized using the Median and a spot intensity [SI1:SI2] data filter was applied to eliminate some of the noisy data. Each spot's color represents a p-value in the range indicated in the scale in the left edge of the image. Note that although all spots are assigned a p-Value, many may not be very significant because adequate preprocessing of the data (such as normalization, and low intensity spot removal, etc.). So use this display with care.
rSq=0.974, n=1728, X(mn+-sd)=(4.477+-7.845), Y(mn+-sd)=(12.379+-24.810)The Scatter plots submenu includes:
If Cy3/Cy5 ratio data is being analyzed, then the "HP F1 vs F2 intensity" menu entry becomes
The following figure illustrates some of the scatter plots and zoomed regions using the scroll bars on the horizontal and vertical axes.
Figure 2.4.4.2 Scatter plot of HP-X and HP-Y single sample data. HP-X is C57B6 pregnancy day 13 and HP-Y is pactation day 1. A) An active scatter plot may be generated for the current HP-X and HP-Y samples filtered by "All named genes". B) similar plot for HP-X and HP-Y 'sets' of replicate samples (3 pregnancy and 4 lactation samples in the sets respectively). Clicking on a point in the plot sets the current gene. C) Zoomed up region (of B) at the bottom of the plot showing more detail and filtered by just "All named genes". Zooming is performed by adjusting the X or Y axes limits scroll bars. Note the points enclosed in magenta boxes indicate genes in the E.G.L. gene list.
Figure 2.4.4.2.1 Scatter plot of multiple channel data from a single sample. A) F1 Vs F2 data for a C57B6 pregnancy day 13 sample. B) Cy3 vs Cy5 data for a NCI mAdb mouse array sample. C) Scatter plot of individual Cy3 channels from two different ratio Cy3/Cy5 data hybridized samples. C) Scatter plot of individual Cy3 channel of HP-X compared with Cy3 channel of HP-Y for ratio Cy3/Cy5 data hybridized samples. D) Scatter plot of individual Cy3 channel of HP-X compared with Cy5 channel of HP-Y for ratio Cy3/Cy5 data hybridized samples.
The Intensity selection plots a histogram of the gene intensity data values for each Filtered spot (gene) in the current hybridized sample.
The Histograms submenu includes:
If Cy3/Cy5 ratio data is being analyzed, then the F1F2 histogram menu entry becomes
The following figures illustrates the histograms. You may use the histogram to specify ranges of [I1:I2] or {R1:R2] for data filtering in the histogram by specifying the corresponding histogram bins. This is described in the figure legend.
Figure 2.4.4.3 Histogram plots. A) Ratio histogram of HP-X/HP-Y data with particular histogram bin selected with the constraint set to filter all genes > that bin. HP-X is 13 day pregnancy C57B6 and HP-Y is day 1 lactatation. The selected bin thresholds are then used in the Filter with the resulting Filtered genes shown in the array image. B) Zdiff histogram of HP-X - HP-Y 'sets' for same data as (A) but with the >< threshold constraint set to find genes outside of the symmetric histogram range. C) Intensity histogram of HP-X data filtered by [I1:I2] intensity range. As with ratio histograms, you can do additional filtering by selecting a particular histogram bin that is then used in the Filter. Filtering was disabled for the intensity histogram. To apply the filter, the "Don't re-Filter" button would be toggled to the "Re-Filter" state. The threshold constraints include: =, >, <, >, <>, and ><. Note that each time you click on the "Thr:" button, it cycles to the next option in the threshold constraints list.
You many generate as many individual expression profile plots as you want using the Display a gene's expr. profile for HP-E command. However, only the last one will be active and will be updated with different genes as you click on them in the microarray image scatter plot. This could be used to compare the EP plots for several different genes. First view the EP plot for one gene, then create a new EP plot for the second gene, etc.
If you use the Display Filtered genes expr. profiles for HP-E command, it will generate a scrollable list of expression profile plots for all of the genes passing the Filter. If the number of genes is very large, it may take a while.
You may interrogate a line corresponding to a particular HP sample in a EP plot by moving the mouse over the line and then selecting the line. This will cause the name of the HP, its intensity and CV to appear in the plot. If the Err check box is set, then the mean of the intensity is indicated by a short horizontal bar and the +- CV by red vertical error bars above and below the mean. If the plot style Line button is pressed, then the plot style is cycled between Line (vertical lines for each point), Circle (small circles at each point), and Curve (circles are connected). Pressing the button repeatedly cycles through: Line (i.e. vertical vars), Circle, or Curve (i.e. continuous curve of all samples). In the case of mean expression profiles used in K-means clustering, the standard deviation is used in place of the CV value. The various clustering methods have EP plots buttons. When they are invoked, the scrollable list of EP plots is sorted by the clustering method ordered list of genes. This enables you to view the data in the same order as that produced by the cluster analysis. If the zoom nnX button is pressed, then all of the plots are magnified by nn-fold to make low intensity plots more visible. Pressing the button repeatedly cycles through: 1X, 2X, 5X, 10X and 20X. It does not change the data itself. The Show HP names button pops up a numbered list of all HP entries used in the expression profile. If you are in stand-alone mode, a SaveAs GIF button will also be available for the EP overlay mode (Figure 2.4.4.4.1) or individual EP plot. This saves the current plot as a full resolution GIF file specified by the user in a popup file browser window.
The Expression profile plots submenu contains:
Use EP overlay else EP list [CB] - display Filtered genes
as an overlay plot of expression profiles, else as a scrollable
list of EP plots.
Figure 2.4.4.4 Expression profile plots. A) Individual expression profile plots may be created by clicking on any gene. Multiple instances may also be created. Here we show some of the presentation options for the 38 sample MGAP database. Error bars are computed for the standard error for that sample. There are three different plotting options: line, circle and curve. #1 is the default line plot with error bars. #2 is the line plot without the error bars but clicking on line 7 to find out which sample it is and what the intensity value is. #3 is the circle plot with error bars, and #4 is the curve plot without error bars. Window #5 shows the list of samples corresponding to the 38 points in the EP plots. B) List of EPplots of the oncogenes and proto-oncogenes in the database (set by the guesser with "onco" and "Set E.G.L." and the Edited Gene List Filter). The list would become scrollable if there were more than 10 profiles. Setting the current gene would scroll the list to the EPplot for the current gene.
Figure 2.4.4.4.1 Expression profile plots. A) Scrollable list of EP plots of Filtered named genes centered at Carbonic anhydrase III. B) Overlay plot of all named Filtered genes. C) Overlay plot of all ONCO or PROTO-ONCO genes with the draw EGL option active so the graphs are drawn for these genes.
