Figure 2.4.3 Filter menu. The Filter menu is a cascade of data filters that restrict the set of genes passing all filters that have been enabled and whatever the criteria was that was set for those filters. This figure shows the GeneClass filter set to "All genes and ESTs", the spot CV filter and Ratio (X/Y) range filters being set interactively by the scroll bars on the right. The genes that pass the filter are indicated with a red (white) circle in the array intensity (ratio) pseudoarray image.

• Filter by GeneClass membership [CB] - only include genes that are members of the current GeneClass.
• Filter by 'User Filter Gene Set' membership [CB] - only include genes that are members of the current 'User Filter Gene Set'.
• Filter by 'Edited Genes List' membership [CB] - only include genes that are members of the 'Edited Gene List'.
• Filter by global 'Good Genes List' membership [CB] - only include genes that are members of the list of good genes. [These genes are indentified by a QualCheck entry in the GIPO database file.]
• Filter by 'Genes with replicates' [CB] - only include genes that genes that have at least 2 copies of the gene replicated on the array. Note: duplicated genes (i.e. F1, F2, etc) are not considered replicates for this purpose.
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• Filter by ratio or Zdiff histogram bin [CB] - only include genes that are in the range of the ratio or Zdiff histogram bin you have clicked on (should be set from histogram plot, but may be turned off here)
• Filter by intensity or (Cy3/Cy5) histogram bin [CB] - only include genes that are in the range of the intensity histogram bin you have clicked on (should be set from histogram plot, but may be turned off here)
• Filter by positive intensity data - filter by positive intensity data if the data may contain negative numbers. Otherwise it will use both positive and negative data. If the database has 2 channels (F1, F2) or (Cy3,Cy5) each channel is checked. If the background correction is enabled, the background corrected values are tested to see if any of them are negative.
• Filter by genes with non-zero intensity [CB] - only include genes that have non-zero density. This protects against zero data that may be present in the database when taking logs of the data.
• Filter by per-sample Good Spot data - filter out genes that do not have "Good Spot" values (defined by the optional QualCheck spot data on a per-sample (i.e. HP) basis. See the list of codes in Appendix C.4). If there is no such spot quality data, then all spots are considered "good".
• Filter by per-sample Spot Detection Value data - filter out genes that do not have "Detection Value" values (defined by the optional DetValue or CorrCoef spot data on a per-sample (i.e. HP) basis. Typical Detection Values could be the Affymetrix MAS5.0 "Detection p-value" or other continuous value of spot detection quality.
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• Filter by spot intensity [SI1:SI2] sliders - filter by individual spot intensity (Cy3 and Cy5 channels if ratio data) within [SI1:SI2] threshold ranges
• Filter by [I1:I2] sliders - filter by gene expression (or Cy3/Cy5 if ratio data) within [I1:I2] threshold ranges
• Filter by ratio or Zdiff sliders - filter by gene ratios or Zdiff values within [R1:R2] or [Z1:Z2] threshold ranges (depending on the normalization method)
• Filter by Cy3/Cy5 HP-X ratio or Zdiff sliders - filter by gene ratios or Zdiff values within [CR1:CR2] or [CZ1:CZ2] threshold ranges (depending on the normalization method). This is useful for filtering data from a single sample.
• Filter by spot CV - filter out genes that do not meet minimum Coefficient of Variation (CV) values of spot replicates (F1 and F2 for the same HP, replicates in HP-X and HP-Y 'sets' of samples etc.).
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• Filter by HP-X,HP-Y t-Test [p-value] slider [RB] - only include genes that meet the HP-X,HP-Y t-Test criteria if they have (F1,F2) duplicate spot (this is a weak form of the t-Test).
• Filter by HP-X,HP-Y 'sets' t-Test [p-value] slider [RB] - only include genes that meet the HP-X,HP-Y 'sets' t-Test criteria (only works if using HP-X and HP-Y 'sets' mode where there are replicate samples).
• Filter by HP-X,HP-Y 'sets' Kolmogorov-Smirnov test [p-value] slider [RB] - only include genes that meet the HP-X,HP-Y 'sets' KS-Test criteria (only works if using HP-X and HP-Y 'sets' mode where there are replicate samples).
• Filter by current Ordered Condition List (OCL) F-Test [p-Value] slider [RB] - only include genes that meet the F-test criteria on the current OCL. This only works if there are at least 2 (replicate) samples/condition for each of the condition sets in the OCL. See info on defining the OCL and using the OCL data.
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• Filter by HP-E clustering [Cluster dist] slider [CB] - only include genes that meet the clustering criteria (alternatively, see the Cluster menu commands).
• Filter by Diff(HP-X,HP-Y) [Abs.Diff.] slider [CB] - only include genes whose absolute difference between mean HP-X and HP-Y (single or 'sets') is < threshold.
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• Filter N genes with highest X/Y ratio or X-Y Zdiff [CB] - look at highest ratios or Zdiff values. The value of N is set in the Edit menu preferences.
• Filter N genes with lowest X/Y ratio or X-Y Zdiff [CB] - look at lowest ratios. The value of N is set in preferences. N is set in the Edit menu preferences.

The Filter by positive intensity data submenu filter contains options that specify which spot intensity values are to be considered when excluding negative quantified spot data. Note: this filter only makes sense if your data might have negative values (e.g. Affymetrix chip "Avg Diff" data) or a background corrected value that is less than 0.0. The filter is enabled by setting the "Filter by spots with positive intensity" checkbox. Negative intensity values may occur with some types of arrays quantification programs. In the "Check spots for positive values mode" submenu, you may set the samples where the test may be applied to spots from the current HP, the single (HP-X,HP-Y) samples, (HP-X,HP-Y) 'sets' (replicated spots), or samples in the HP-E list selected to be used in the filter. If there are (F1,F2) or (Cy3/Cy5) data, then each spot must meet the threshold criteria.

• Current HP [RB] - spots in current sample spots
• HP-X & HP-Y [RB] - spots in X and Y single samples
• HP-X or HP-Y 'sets' [RB] - spots in the HP-X set or HP-Y set
• HP-X & HP-Y 'sets' [RB] - spots in both the HP-X set and HP-Y set
• HP-E [RB] - spots in HPs in expression profile list

The Filter by Good Spot data submenu filter contains options that specify spots based on their quality. It filters out genes that have that do not have "Good Spot" values defined by the optional QualCheck spot data. (See the list of codes in Appendix C.4). If there is no such spot quality data, then all spots are considered "good". The filter is enabled by setting the "Filter by spots with Good Spot values" checkbox. All spots for the specified samples must meet the criteria. In the "Check spots for Good Spot mode" submenu, you may set the samples where the test may be applied to spots from the current HP, the single (HP-X,HP-Y) samples, (HP-X,HP-Y) 'sets' (replicated spots), or samples in the HP-E list selected to be used in the filter.

• Current HP [RB] - spots in current sample spots
• HP-X and HP-Y [RB] - spots in X and Y single samples
• HP-X or HP-Y 'sets' [RB] - spots in HP-X set or HP-Y set
• HP-X and HP-Y 'sets' [RB] - spots in HP-X set and HP-Y set
• HP-E [RB] - spots in HPs in expression profile list

The Filter by Spot Detection Value data submenu filter contains options that specify spots based on their spot detection value quality metric over the range of [0.0 : 1.0]. The filter is available only if the data exists for your database and is ignored otherwise. If active, it pops up a "Spot Detection Value" slider in the range of [0.0 : 1.0]. Only spots greater than the slider value pass the filter. This data could be the Affymetrix MAS5.0 "Detection p-value" or some other metric correlated with spot detection quality. The filter is enabled by setting the "Filter by per-sample Spot Detection Value" checkbox. All spots for the specified samples must meet the criteria. In the "Check spots for Spot Detection Value mode" submenu, you may set the samples where the test may be applied to spots from the current HP, the single (HP-X,HP-Y) samples, (HP-X,HP-Y) 'sets' (replicated spots), or samples in the HP-E list selected to be used in the filter.

• Current HP [RB] - spots in current sample spots
• HP-X and HP-Y [RB] - spots in X and Y single samples
• HP-X or HP-Y 'sets' [RB] - spots in HP-X set or HP-Y set
• HP-X and HP-Y 'sets' [RB] - spots in HP-X set and HP-Y set
• HP-E [RB] - spots in HPs in expression profile list

The Filter by spot intensity [SI1:SI2] sliders submenu contains options that determines how individual spot intensity thresholding is to be applied in the Filter.

• Use spot intensity [SI1:SI2] sliders [CB] - use spot intensity thresholding
• Inside [RB] - test inside of [SI1:SI2] range
• Outside [RB] - test outside of [SI1:SI2] range
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• Use data mode - specify which samples are tested
• Compare channels meeting range - specify which additional constraints are used. This is useful for finding genes with high or low expression but that has some samples that have opposite expression.

The Use data mode submenu filter contains options that specify which spot intensity values are to be considered of the single sample (F1 and F2 replicated spot intensity data, or Cy3/Cy5 for ratio data), or the (HP-X,HP-Y) 'sets' of replicated samples is to be used in the filter. If there are single sample (F1,F2) or (Cy3/Cy5) data, then each spot must meet the threshold criteria.

• Current HP [RB] - spots in current sample spots
• HP-X & HP-Y [RB] - spots in X and Y single samples
• HP-X & HP-Y 'sets' [RB] - spots in HP-X set and HP-Y set
• HP-E [RB] - spots in HPs in expression profile list

The Compare channels meeting range submenu specifies which additional constraints are to be used. If required by the (AT MOST channels, AT LEAST channels, PRODUCT OF channels, SUM OF channels) commands, the Percent SI OK scroll bar will appear which covers the range of 0% to 100%.

• ALL channels [RB] - ALL channels must meet the range specification
• ANY channels [RB] - ANY channels may meet the range specification
• AT MOST channels [RB] - AT MOST Percent SI OK channels may meet the range specification
• AT LEAST channels [RB] - AT LEAST Percent SI OK channels may meet the range specification
• PRODUCT of channels [RB] - the PRODUCT of all channels must meet the range specification
• SUM of channels [RB] - the SUM of all channels must meet the range specification

The Filter by [I1:I2] sliders submenu contains options that determines how spot expression (intensity or (Cy3/Cy5) ratio value) thresholding is to be applied in the Filter:

• Use intensity [I1:I2] sliders [CB] - use spot intensity thresholds I1 (lower) and I2 (upper)
• Inside [RB] - test for intensity inside of [I1:I2]
• Outside [RB] - test for intensity outside of [I1:I2]

The Filter by ratio or Zdiff sliders submenu contains options that determines how spot-ratio thresholding is to be applied in the Filter. The spot ratio is mean HP-X / mean HP-Y for sets of samples. The spot Zdiff is used if one of the Zscore normalization methods is active and is computed as (mean HP-X - mean HP-Y) for sets of samples.

• Use ratio [R1:R2] or Zdiff [Z1:Z2] sliders [CB] - use spot ratio [R1:R2] or Zdiff [Z1:Z2] range thresholds
• Inside [RB] - test inside of [R1:R2] or [Z1:Z2] range
• Outside [RB] - test outside of [R1:R2] or [Z1:Z2] range

The Filter by Cy3/Cy5 HP-X ratio or Zdiff sliders submenu contains options that determines how spot Cy3/Cy5 HP-X ratio thresholding is to be applied in the Filter. The spot ratio is Cy3/Cy5 for normalized data unless one of the Zscore methods is used. In that case, the Zdiff is used and is computed as (Cy3 - Cy5) for sets of samples. If HP-X 'sets' is used, then it computes the mean Cy3 value and the mean Cy5 value and uses those values in the above computations.

• Use ratio [R1:R2] or Zdiff [Z1:Z2] sliders [CB] - use spot ratio [R1:R2] or Zdiff [Z1:Z2] range thresholds
• Inside [RB] - test inside of [R1:R2] or [Z1:Z2] range
• Outside [RB] - test outside of [R1:R2] or [Z1:Z2] range

The Filter by spot CV submenu filter contains options that specify how the Coefficient Of Variation of the (F1,F2) or (HP-X,HP-Y) 'sets' (replicated spots) is to be used in the filter. The (F1,F2) CV is available only if there are duplicate spots on the HPs.

• Use spot [CV] slider [CB] - apply one of the spot CV filter modes as a Filter and popup a CV slider to set the threshold
• CV spot filter mode - select samples to be used in computing the CV
• Use mean else max of CVs [CB] - compute the CV as the maximum or the mean of the CVs of the samples selected

Filtering using statistical test by your selecting a p-value

Filter by current Ordered Condition List (OCL) F-Test [p-Value] slider [RB] - only include genes that meet the F-test criteria on the current OCL. This only works if there are at least 2 (replicate) samples/condition for each of the condition sets in the OCL. See info on defining the OCL and using the OCL data.

Filtering out genes with high replicate spot variation

The Spot CV filter mode submenu contains options to select how the spot CV filter is to be applied. It computes the maximum value of CV for all of the samples in the particular sample set specified. That maximum value is then used for the spot CV filter test. Genes may be filtered out having a large difference between spot quantification values of corresponding duplicate spots. You may compute the coefficient of variation CV_j for the two values (f1_j and f2_j for a particular gene j.

    CVj = 2|f1j-f2j|/(f1j+f2j)

If the database only has one field but replicate HPs, then you may use the HP-X & HP-Y 'sets' CV_j to filter the genes. Then CV_j values are tested against a CV threshold slider value to eliminate genes with a high coefficient of variation.

• Current HP [RB] - CV of (F1,F2) for each gene in current sample [if duplicate spots are available on each sample]
• HP-X or HP-Y [RB] - CV of (F1,F2) for HP-X and HP-Y single samples [if duplicate spots are available on each sample]
• HP-X 'set' [RB] - CV of spots in HP-X set
• HP-Y 'set' [RB] - CV of spots in HP-Y set
• HP-X or HP-Y 'sets' [RB] - CV of spots in the HP-X set or HP-Y set
• HP-X and HP-Y 'sets' [RB] - CV of spots in both the HP-X set and HP-Y set
• HP-E [RB] - CV of HPs in expression profile list

2.4.3.1 Data filtering using multiple gene data filters

Any or all of the data filters may be selected simultaneously. In particular, if you select filters that use parameter threshold scrollers, they will be added to a state scroller window (see Figure 2.3.4.1 for details to allow adjustment of ALL sliders simultaneously). You may change various thresholds and see the effect in real time. Note: some of the scrollers are more sensitive to low values. Therefore, we set them to respond non-linearly with a more precise vernier at the low end.

Figure 2.4.3.1 Filtering using multiple scrollers. This example is of Cy3/Cy5 time series data. It filters normalized spot intensity of the Cy3 and Cy5 channels independently ([SI1:SI2] inside range) where low intensity spots are eliminated. It then filters out genes outside of the [R1:R2] ratio range.

Figure 2.4.3.2 Using the Positive Intensity data Filter. This allows removing negative data if the data contains negative intensity values (e.g. Some Affymetrix data has negative Average Difference values which could be read as Intensity for MAExplorer).

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