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#63 Handling of 4 color GenePix 4 gpr, jpegs
GenePix 4 allows 4 color images. This brings up a number of
issues. I'll review what I learned about how GP4 handles this, and
then bring up how we might address this.
What GenePix 4 spits out when you save a results file (4 color
mode):
(Name stands for the name that you designate for the files when
you execute the Save Results As
option.)
7 jpegs:
3 of these have images for the pseudocolored ratios:
Name_R1
Name_R2
Name_R3
Note that in GenePix you have the option of choosing up to 3
ratios (e.g., color1 vs 2, 1 vs 3, and 2 vs 4). You can choose just
1 or 2 ratios (or 0?). Im guessing that the _R2 and _R3 files are
only generated if youve selected a second and third ratio.
4 of these are single colors (wavelengths):
Name_W1
Name_W2
Name_W3
Name_W4
Again, I assume that if you only load 3 colors you dont get a _W4
file and so on.
The final file generated is (of course) the gpr file. Brian asked
about how the normalizations are handled. Heres what I deduce
from inspecting the files. In GP4, one chooses a normalization
scheme and can then apply it. (It can be removed later.) If the
normalization is not applied when the gpr is saved, then the
header indicates that:
"NormalizationMethod=None"
"NormalizationFactors=1 1 1 1"
The factors are tab delimited.
If the normalization has been applied, the header states:
"NormalizationMethod=
RatioOfMedians,AllFeatures,MeanValue:1.000000"
"NormalizationFactors=2.1586 2.1586 0.463263 0.463263"
This example shows the default method. Obviously there are other
normalization method choices as well. 4 normalization factors are
given (one for each wavelength). The only reason that the first 2
normalization factors are the same and the last two are the same
is that for this example I used one image for both wavelengths 1
and 2 and another for both 3 and 4. All intensity values
(foregrounds and backgrounds) are multiplied by the normalization
factor for that wavelength. So if the non-normalized value for F635
Median was 200, the B635 Median was 100, and the normalization
factor for that wavelength (635) is 2.1586, then the normalized
values reported in the gpr file would be 432 for F635 Median and
216 for B635 Median.
Note also that the gpr file reports only the 3 ratios requested by
the user. There are 6 possible ratios (actually 12 if you count W1/
W2 and W2/W1 separately, as GenePix does). This means that
the some ratios that might be of interest arent reported in the gpr
file. We could have NOMAD calculate them from the raw data.
All this brings up an issue I havent thought about before: with >2
colors, do you normalize each color or do you normalize by pairs
of colors (i.e., for each of the 6 possible ratios)?
Questions:
1. Does NOMAD deal with the variable number of jpeg files
produced by GP4 now?
2. Which GP4 jpeg files should NOMAD upload? Right now for
GP3 only one jpeg is uploaded this corresponds to the _R1 file
in GP4. Should we upload all the ratio files (up to 3 for GP4)?
Since some ratios arent included, should we upload the individual
wavelengths (_W1-_W4 for a 4 color array) in addition or instead of
the _R jpegs?
3. How will NOMAD do its own normalizations for GP4 data, esp.
GP4 files that have >2 colors. Will it normalize by multiplying
each wavelength by a factor (like GP4 does itself) or will it
normalize each possible ratio separately? The first method seems
easy but probably wrong. The second method will be more
computationally intensive but I suspect more satisfactory.
As none of us are using >2 colors yet, I dont see that we need to
solve most of this for 0.8.5.
I append the header and the first few lines of data from a 4 color
GP4 gpr file. (I didnt have a real gal file for this so the probes are
nameless
)
ATF 1.0
27 82
"Type=GenePix Results 2"
"DateTime=2001/02/14 13:54:00"
"Settings="
"GalFile="
"PixelSize=10"
"Wavelengths=635 488 532 350"
"ImageFiles=C:\Documents and Settings\derle\Desktop\test635.tif
0 C:\Documents and Settings\derle\Desktop\test488.tif 0 C:\
Documents and Settings\derle\Desktop\test532.tif 0 C:\
Documents and Settings\derle\Desktop\test350.tif 0"
"NormalizationMethod=None"
"NormalizationFactors=1 1 1 1"
"JpegImage=C:\Documents and Settings\derle\Desktop\
test_no_norm.jpg"
"RatioFormulations=W1/W2 (635/488) W1/W3 (635/532) W1/
W4 (635/350)"
"Barcode="
"BackgroundSubtraction=LocalFeature"
"ImageOrigin=0, 0"
"JpegOrigin=1040, 2280"
"Creator=GenePix Pro 4.0.1.9"
"Scanner="
"FocusPosition=0"
"Temperature=0"
"LinesAveraged=1"
"Comment="
"PMTGain=0 0 0 0"
"ScanPower=100 100 100 100"
"LaserPower=0 0 0 0"
"LaserOnTime=0 0 0 0"
"ScanRegion=0,0,1745,2182"
"Supplier="
"Block" "Column" "Row" "Name" "ID" "X" "Y" "Dia."
"F635 Median" "F635 Mean" "F635 SD" "B635 Median"
"B635 Mean" "B635 SD" "% > B635+1SD" "% > B635+
2SD" "F635 % Sat." "F488 Median" "F488 Mean" "F488
SD" "B488 Median" "B488 Mean" "B488 SD" "% > B488+
1SD" "% > B488+2SD" "F488 % Sat." "F532 Median"
"F532 Mean" "F532 SD" "B532 Median" "B532 Mean"
"B532 SD" "% > B532+1SD" "% > B532+2SD" "F532 %
Sat." "F350 Median" "F350 Mean" "F350 SD" "B350
Median" "B350 Mean" "B350 SD" "% > B350+1SD" "% >
B350+2SD" "F350 % Sat." "Ratio of Medians (635/488)"
"Ratio of Means (635/488)" "Median of Ratios (635/488)"
"Mean of Ratios (635/488)" "Ratios SD (635/488)" "Rgn Ratio
(635/488)" "Rgn R_ (635/488)" "Ratio of Medians (635/532)"
"Ratio of Means (635/532)" "Median of Ratios (635/532)"
"Mean of Ratios (635/532)" "Ratios SD (635/532)" "Rgn Ratio
(635/532)" "Rgn R_ (635/532)" "Ratio of Medians (635/350)"
"Ratio of Means (635/350)" "Median of Ratios (635/350)"
"Mean of Ratios (635/350)" "Ratios SD (635/350)" "Rgn Ratio
(635/350)" "Rgn R_ (635/350)" "F Pixels" "B Pixels" "Sum
of Medians" "Sum of Means" "Log Ratio (635/488)" "Log
Ratio (635/532)" "Log Ratio (635/350)" "F635 Median - B635"
"F488 Median - B488" "F532 Median - B532" "F350 Median -
B350" "F635 Mean - B635" "F488 Mean - B488" "F532
Mean - B532" "F350 Mean - B350" "Flags" "Normalize"
1 1 1 "" "" 1290 2560 120 253 229 55 92
93 4 100 100 0 253 229 55 92 93 4 100
100 0 1174 1044 329 203 210 37 100 100 0
1174 1044 329 203 210 37 100 100 0 1.000
1.000 1.000 1.000 1.000 1.000 1.000 0.166 0.163
0.167 0.162 1.142 0.164 0.983 0.166 0.163 0.167
0.162 1.142 0.164 0.983 120 630 2264 1956
0.000 -2.592 -2.592 161 161 971 971 137 137
841 841 0 1
1 2 1 "" "" 1470 2560 100 92 92 3 95 96
6 2 0 0 92 92 3 95 96 6 2 0 0 198 200
15 218 228 41 0 0 0 198 200 15 218 228
41 0 0 0 1.000 1.000 1.000 1.000 1.000 1.000
1.000 0.150 0.167 0.156 0.175 2.198 0.112 0.438
0.150 0.167 0.156 0.175 2.198 0.112 0.438 80
448 -46 -42 0.000 -2.737 -2.737 -3 -3 -20 -20 -
3 -3 -18 -18 -50 0
1 3 1 "" "" 1660 2550 90 266 332 215 96
97 7 90 84 0 266 332 215 96 97 7 90 84
0 1550 2111 1835 221 232 53 86 75 0 1550
2111 1835 221 232 53 86 75 0 1.000 1.000
1.000 1.000 1.000 1.000 1.000 0.128 0.125 0.142
0.153 1.488 0.120 0.973 0.128 0.125 0.142 0.153
1.488 0.120 0.973 52 385 2998 4252 0.000 -2.967
-2.967 170 170 1329 1329 236 236 1890 1890 0
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