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This quick analysis manual explains how to perform a simple analysis using the Coffalyser VBA analysis
software V8. This manual is an extension on the normal complete manual from v7 and only explains how
to perform a quick analysis. A complete overview on support documents can be found at
http://www.mlpa.com/coffalyser and in the document available online
How_to_use_the_Coffalyser_Support_Page_v1.
CONTENT
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
3.
4. Changing the Actual length of the control fragments for automated quality control
A deviation between the cloned real fragment lengths and the actually detected fragment
lengths always exist. To enable the automated quality control of the Coffalyser the actual
lengths of the control fragments need to be adapted before data filtering. After opening the
Coffalyser user form, click in the All MLPA mixes screen on control and mut probes on the left
side of the screen. (You may navigate between the different screens by clicking on the tabs on
the topside of the program. The tab surrounded with the pink edge is the active screen. ) A list
with all control fragments will appear as well as (if present) mutation specific probes (figure 4).
You need to note the actual lengths of the control fragments and mutation specific probes in the
size called electropherogram (figure 3) and then adjust these lengths in the coffalyser before
data filtering. The concentration control fragments (Q-fragments) are expected at 61, 68, 74
and 80 bp and may only be visible in a NO-DNA run. Double click on each of the listed lengths
and change these to the actually detected length. Repeat this procedure for the denaturation
fragements (D-fragments) which are expected at 88 and 96 bp and for the Y and X specific
fragments which are expected at resp. 100 and 105 bp. After finishing click on file and select
Save all mix changes, overwrite the old file.
If these fragments are not in your kit, then you dont have to change the lengths and you may
ignore the possible warnings (e.g. incomplete denaturation).
158.42
153.37
193.05
336.20
80000
163. 87
85.55
135.01
70000
316. 97
354.06
346.33
245. 29
95.98
221.23
229.02
60000
209. 72
201.60
105. 66
91.17
50000
310.89
264.28
183.22
177.59
426.74
301. 47
436.64
291. 34
141. 28
253.82
40000
397.22
466. 57
455.58
100. 80
30000
415. 62
390.98
383.75
282.57
374. 74
409. 00
475.54
445.17
20000
Dye Signal
79. 49
73.30
482.77
10000
0
50
100
150
200
250
Size (nt)
300
350
400
450
Single sample files created in Genemapper software by export of a single size called sample file
"Import GENEMAPPER TXT FILE (multiple runs)"
Mulitple runs files created in genemapper software exported in the plot view when displaying
multiple runs
"Import PEAK SCANNER FILE (combined table)"
Multiple runs file created in Peak Scanner Software after size calling
"Import SPECTROPHOTOMETRIX FILE (combined table)"
Multiple runs file created using the export txt file option the spectrometrix software data
"Import Megabace file (combined table)"
Multiple runs file created using the export txt file option after size calling in the Megabace
software
"Import LICOR file (GeneTools)"
Multiple runs file created using by Genetools scanning a slabgel image of a licor device
"Import GENESCAN TXT FILE (single)"
Single sample files created in Genescan software by export of a single size called sample file
"Import GENESCAN PROJECT FILE (multiple runs)"
Mulitple runs files created in Genescan software exported in the plot view when displaying
multiple runs
"Import ABI -310(0)/3700 FSA FILES"
Size caling of raw fsa files coming from the older series of ABI may be performed directly. These
files will be size called and transformed to genescan txt files during import.
low background. Next click on AddA (>>) while the combo box on the left side is set to
add/remove reference runs. The names will move underneath the header Reference Runs,
now change the combo box on the left side to perform autobin on references (P) and click on
the left play button.
The program will now try to find the actual length belonging to the probes in the mix. After
finishing the actual lengths will be displayed underneath SET BIN while the cloned lengths of
the probes can be seen underneath CLONED LENGTH. Check the estimated lengths underneath
SET BIN with for instance an image of an size called electropherogram and make sure that they
all contain valid values. Alternatively you may double click on a run name in the left column. All
fragments lengths and intensities of the set metric will appear in the most right columns. You
can use this list to define the actual length if they were not estimated correct. Probe signals will
always be close to the cloned length and will have high signals intensities as compared to
background signals. Each set bin value can be adapted manually by clicking on the fragment, and
adjusting the value using the pop up box.
When you are sure all actual set lengths in the SET BIN list are correct, change the left combo
box to Save all mix changes and click on the left Play button. Overwrite the old files. The actual
lengths are now set and saved and can be used every time you use this MLPA mix as long as the
capillary device, size calling method, gel, size standard etc remain the same.
To remove imported runs, change the combo box on the left side to Delete imported Files (P)
and click on the left play button.
Click on the Play button on the right side to start data filtering. The program will work of both
lists, when data filtering is finished the quality overview screen should appear.
9. Filtering data for a methylations status analysis
Separate rules apply when you want to perform a methylation status analysis. First of all, you
need to make sure that your cut and uncut runs have equal names to make automatic
comparison possible. A cut run may for instance be named: Sample1-cut, while the uncut run
should then be named: Sample1-uncut. Also see the section on Adjusting File names for this.
Navigate to the data filtering page (figure 7). Next select the all uncut runs in the left column
and click on AddA (>>) while the combobox on the left side is set to add/remove reference
runs. The names will move underneath the header Reference Runs. Next change the
combobox on the left side to add/remove sample runs. Select the CUT runs belonging to your
previously imported uncut runs and click on click on AddA (>>). Your reference and sample data
should now be right underneath the headers REFERENCE RUNS and resp. SAMPLE RUNS. Click
on the play button on the right side to start filtering your data.
10. Checking my imported signals.
Direct after analysis the quality control is opened giving an overview of the found signals and
results of the quality control steps of all imported runs (figure 9). If this menu doesnt open, you
may click on All run QC in the left window. The displayed parameters are: Run name, DNA
concentration estimation, Number of probe signals found, Number of reference probes found,
Ligation fragment found, Male/Female determination (if Y fragment present), Denaturation
check on fragments 88 bp and denaturation check on fragments 96 bp. Please note that these
tests are dependent on the correct settings of the actual lengths before data filtering! When the
probe or control fragments lengths are not set correct, probe signals may be missing or runs
may seem to be denatured incomplete while this is not the case. In doubt always check your
runs on the RAW electropherogram.
To separately remove bad reference or sample runs, first click on Reference runs QC or Sample
runs QC. The same check appears but now only for the reference or sample runs. To prevent
errors during analysis, remove reference runs missing any probe signal and remove sample runs
missing reference probe signals. You may do this by double clicking on the run name in the
center windows while the Reference runs QC or Sample runs QC is displayed.
If you think that some probes were not filtered correct, possible because they are missing in
several runs, click on Reference runs or Sample runs in the left column in the bottom. This will
display the imported signals of each reference or sample run, allowing you to recognize the
probes which has been filtered wrong. You need to go one step back and manually adjust the
set bin for this fragment by double clicking on it.
"Directanalysis"
This method is the most straight forward method to normalize your data. It doesnt correct for
sloping effects. This method should thus only be used when size to signal sloping is more or less
the same between your reference and sample runs. You can always check if there is a sloping
artifact in your results by ordering your ratio results on probe length (after analysis, using the
normal XLS order function) and this may thus be the best method to start with. When the
average ratio of the shorter probes is higher or lower than the average ratio of the longer probes
(not taking into account ratio caused by aberrant genes), you may need to re-perform the
analysis using an alternative analysis method.
This method assumes that the set reference probes remain normal between reference and
sample runs. Each reference probes will be used to separately determine the ratio of a target
probe. The median of all these calculated ratios will estimate the final ratio. The median is the
center value, and this method is thus robust for aberrations in the reference probes as long as
more than 51% of all reference probes remain normal.
"Directmethylation status"
This is almost equal to the directanalysis method in that it doesnt correct for sloping effects. This
will usually not be necessary to perform the methylation status analysis because the same DNA is
being compared to each other (Cut & Uncut). This method assumes the number of changes in the
target sequences of the reference probes is minimal. Each reference probe will be used to
separately determine the ratio of each target probe. The median of all these calculated ratios will
estimate the final ratio. The median is the center value, and this method is thus robust for
aberrations in the reference probes as long as more than 51% of these probes remain normal.
Please note that in many cases some reference probes may give higher or lower signals in the cut
sample. This is not due to aberrations but rather because of the structural changes present after
cutting the DNA or because of the reduced presence of amplifiable targets during the PCR
reaction.
This method calculates the relative ratio of each probe signal between the cut and uncut
reaction. When a target is completely methylated, no target is cut and the signal of this probe is
expected to be ratio 1 or 100%. When a target is hemi-methylated, a single allele is expected to
be completely cut and the expected ratio between the cut and uncut single is 0.5 or 50%.
these regions in the used samples (please check the probe mix description for the targets of the
reference probes in your kit).
The control probes method (LS) first defines if there are outliers between the set reference
probes in each sample assuming that a median of all reference probes is normal. The reference
probes signals that are not outliers are then used to calculate the amount of regression in that
run using an adapted the least of squares regression method. The size to signal drop is thus
expected to be more or less linear, if this is not the case then use one of the LMS methods. Next
to this, the least of squares method is sensitive with a low number of signals. It may thus be
recommended to use the Tumor analysis (LS) method instead which is more robust and also
normalizes against the reference probes. After regression correction the method assumes that
the set reference probes remain normal between reference and sample runs. Each reference
probes will be used to separately determine the ratio of a target probe. The median of all these
calculated ratios will estimate the final ratio. The median is the center value, and this method is
thus robust for aberrations in the reference probes as long as more than 51% of all reference
probes remain normal.
Each target probe will be used to separately determine the ratio of each target probe. The
median of all these calculated ratios will estimate the final ratio. The median is the center value,
and this method is thus robust as long as more than 51% of all probes remain normal.
The regression is determined directly on all reference probe signals (without outlier detection)
using a least of median squares method. This method is very robust in the presence of outliers,
After correction each reference probe will then be used to separately determine the ratio of each
target probe. The median of all these calculated ratios will estimate the final ratio. The median is
the center value, and this method is thus robust for aberrations in the reference probes as long
as more than 51% of these probes remain normal. Please note that in many cases some
reference probes may give higher or lower signals in the cut sample. This is not due to
aberrations but rather because of the structural changes present after cutting the DNA or
because of the reduced presence of amplifiable targets during the PCR reaction.
This method calculates the relative ratio of each probe signal between the cut and uncut
reaction. When a target is completely methylated, no target is cut and the signal of this probe is
expected to be ratio 1 or 100%. When a target is hemi-methylated, a single allele is expected to
be completely cut and the expected ratio between the cut and uncut single is 0.5 or 50%.
"USER DEFINED"
Putting the combobox on user defined means that you may define all steps yourself. You can do
this by clicking on settings and making adjustments at the different tabs. This is only
recommended for the advanced user. More info on advanced settings can be found in the
complete coffalyser manual, appendix Advanced analysis settings.
Bar charts
Click on Result Reports to view the bar charts of your sample runs. In the MLPA results window use the
right combobox to choose the way you want to view your sample results. This is either in a bar ratio chart,
result report method or technical chart. Double click in the left column on a sample name to view the bar
chart. The probes in this chart are ordered in the recommended order, showing the Map view locations
on the X-axis and the found probe ratio on the Y-axis. The reference probes are usually ordered to the
most right position. The error bars coming with the chart are the calculated standard deviations (1x)
found for each probes, assumed you have used reference runs which were performed on normal human
DNA. These standard deviations are calculated from the reproducibility of reference runs and the
confidence of the normalization factor. High standard deviations are usually found when the used
reference samples were not reproducible as compared to each other.
that the normal status during outlier detection may also fall on aberrant probes, e.g. when applying the
tumor analysis method, sloping may be determined on aberrant probes even though the normalization
may normalize the data afterwards to the reference probes.
In figure 12 a sample is visualized analyzed in the Population method. Most probes, including the
reference probes (green dots) give equal signal intensities except for 9 aberrant probes. These 9 aberrant
probes were defined as outliers by dynamic correlation checks (see four outlier lines) and will not be used
for slope correction. The remaining signals will be used for regression correction by calculating a expected
regression line through the remaining point and using the distance of each point to this line for the
coming normalization.
This figure should thus only be used to determine if your sloping correction was ok, and how your
reference probes behave according to each other (in figure 12 they all have the same chromosomal
status, and thus give equal signal intensities, although more or less effected by the sloping artifact).
D1.03.01831DMD__MLPA-P034^GSsTXT.txt
5/2/2008 17:16
Operator
Kit number
User1
R e fe re nc e runs (<7)
45
D1.03.02609DMD__MLPA-P034^GSsTXT.txt
D1.03.02235DMD__MLPA-P034^GSsTXT.txt
YES
Female
0.96 ; 36
0.03 ; 5
OK
Incomplete
Incomplete
Chr pos
Xq11.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp21.2
Xp11
Xp21.2
Xq28
Xq28
Xq28
Length (bp)
138
170
210
242
282
314
354
386
426
458
154
186
226
258
298
330
370
402
442
474
146
178
218
250
290
322
362
394
434
466
162
194
234
266
306
338
378
410
450
482
202
130
490
274
418
MV36
Ratio stdev 95% Range
X-032.0 DMD exon 01
1.05
0.03
1 -1.11
X-032.0 DMD exon 02
1.51
0.05
1.41 -1.61
X-032.0 DMD exon 03
1.48
0.02
1.45 -1.52
X-032.0 DMD exon 04
1.37
0.07
1.24 -1.51
X-032.0 DMD exon 05
1.43
0.07
1.3 -1.56
X-032.0 DMD exon 06
1.41
0.04
1.33 -1.49
X-032.0 DMD exon 07
1.48
0.05
1.37 -1.59
X-032.0 DMD exon 08
1.37
0.03
1.32 -1.43
X-032.0 DMD exon 09
1.41
0.08
1.24 -1.57
X-032.0 DMD exon 10
1.39
0.05
1.3 -1.48
X-032.0 DMD exon 21
1.01
0.03
0.96 -1.07
X-032.0 DMD exon 22
1
0.05
0.91 -1.09
X-032.0 DMD exon 23
0.92
0.01
0.89 -0.94
X-032.0 DMD exon 24
0.95
0.05
0.85 -1.04
X-032.0 DMD exon 25
0.96
0.03
0.9 -1.02
X-032.0 DMD exon 26
0.99
0.03
0.93 -1.06
X-032.0 DMD exon 27
0.94
0.06
0.82 -1.07
X-032.0 DMD exon 28
0.94
0.08
0.79 -1.09
X-032.0 DMD exon 29
1
0.04
0.92 -1.09
X-032.0 DMD exon 30
0.92
0.01
0.91 -0.94
X-032.0 DMD exon 41
1
0.02
0.97 -1.03
X-032.0 DMD exon 42
1
0.03
0.94 -1.05
X-032.0 DMD exon 43
1
0.05
0.91 -1.1
X-032.0 DMD exon 44
1.01
0.04
0.93 -1.1
X-032.0 DMD exon 45
1.02
0.05
0.92 -1.11
X-032.0 DMD exon 46
1.01
0.06
0.89 -1.13
X-032.0 DMD exon 47
0.99
0.11
0.78 -1.2
X-032.0 DMD exon 48
0.96
0.1
0.76 -1.16
X-032.0 DMD exon 49
0.97
0.01
0.95 -0.99
X-032.0 DMD exon 50
1.02
0.1
0.83 -1.21
X-032.0 DMD exon 61
1.02
0.08
0.87 -1.17
X-032.0 DMD exon 62
1.07
0.07
0.94 -1.2
X-032.0 DMD exon 63
1.01
0.08
0.86 -1.16
X-032.0 DMD exon 64
1
0.05
0.9 -1.1
X-032.0 DMD exon 65
1
0.09
0.83 -1.17
X-032.0 DMD exon 66
1.02
0.1
0.82 -1.22
X-032.0 DMD exon 67
1
0.11
0.78 -1.23
X-032.0 DMD exon 68
1.01
0.09
0.83 -1.2
X-032.0 DMD exon 69
0.91
0.07
0.78 -1.04
X-032.0 DMD exon 70
0.94
0.06
0.83 -1.06
c
1.01
0.01
0.99 -1.04
c
1.09
0.02
1.05 -1.12
c
0.98
0.02
0.93 -1.02
c
1
0.03
0.94 -1.06
c
0.96
0.05
0.87 -1.05
Figure 13 MLPA results report image, created by saving result report as image.
G ain?
DEL?
320:1
1:1805
1:62596
1:48
1:192
1:383
1:893
1:220
1:59
1:169
2000:1
467:1
396:1
206:1
956:1
1368:1
103:1
57:1
574:1
555:1
31114:1
3961:1
390:1
524:1
253:1
151:1
29:1
29:1
12851:1
29:1
55:1
31:1
74:1
328:1
56:1
23:1
22:1
36:1
61:1
123:1
25597:1
82:1
3433:1
2189:1
303:1
58570:1
233:1
270:1
396:1
304:1
361:1
256:1
459:1
310:1
404:1
194816:1
34198:1
8189:1
6390:1
41135:1
93238:1
2938:1
1345:1
45293:1
12762:1
2557173:1
295696:1
30069:1
48244:1
23415:1
11746:1
1246:1
852:1
648666:1
2068:1
4416:1
5499:1
5032:1
23714:1
3292:1
1579:1
1053:1
2206:1
1029:1
3566:1
2511315:1
25688:1
183757:1
168249:1
11305:1
14.
Exploring results 2 (No sample reports enabled)
After the analysis is finished, the results page will automatically open (also when no reports are generated). Click
in the right column on Open the quick explorer, the main user form will minimize and the quick results explorer
will open together with the sheets containing the results. You can now use the results explorer to guide you to the
calculated ratio results or just navigate to the different sheets. You can also use the minimize button in the right
top, the coffalyser user form will then close and you can explore your results on the sheets.
To minimize the number of available sheets, most calculation sheet will be hidden if you use the minimize button
or quick explorer option. To view all sheets you can use the right mouse click button and click on Show all Sheets,
you can also use the right mouse button to reopen the coffalyser user form by clicking on Open coffalyser.
Figure 14 Quick results explorer form. Change the combo box to the results type you wish to view and
then select the sample name in the main window. By clicking on Save results summary you can save a
file containing all relevant result sheets.
Ratio result sheet
The most important sheet is probably the ratio results sheets or RESULTS (1), this sheet contains the calculated
ratio results of all analyzed sample runs. Above the calculated ratios sample information and quality check points
are displayed, in order they are: Sample name, analysis data, chosen normalization method, if an average or
median over the reference probes was used as a normalization factor, the number of found probes, the number of
found reference probes, if the ligation control peak (92 bp was found), if the sample was male or female (if a Y
probe was present), the PPMC and MAD values (which will be explained more down, if there was enough DNA
during the MLPA reaction (by estimation of the relative signal of the Q-fragments (60, 68, 74, 80 bp)), if the DNA
was denatured completely (by estimation of the relative signals of the DD fragments (88, 96 bp)).
PPMC
The PPMC value, or Pearson product moment correlation value is the correlation value found when slope
correction is applied. For every MLPA run, first the probe specific biases are corrected; hereafter the signals which
originate from targets with a normal status are expected to have a linear relationship with each other. These
signals either originate from the reference probes (control probe methods) or all probes (tumor analysis /
population method). Whether the signal are normal or not is defined by a outlier detection system which follows a
Monte Carlo like simulation assuming that the median of all signals is the normal status, or can be used to find the
sloping relation. Sloping correction can thus also be done on signals that are actually aberrant, but do show a linear
relationship with all, cause the majority contains the same chromosomal status. In most cases correction of sloping
can best be performed using all signals, this being much more robust that correcting on the reference probes only.
Correction on the reference probes (control probes methods) is thus only recommended when the number of
reference probes is 10 or more.
MAD VALUE
The MAD value or median of absolute deviation is the value which arises when you subtract the final estimated
ratio (displayed in RESULTS (1)) from each separately calculated probe ratio (using each reference probes
separately) and then take the median value of these. This value is thus very small (or zero) if each reference probes
creates the same result, if this is not the case the target sequences to which the reference probes hybridize may be
aberrant. The median estimator is however quite robust though a higher MAD value may sincerely compromise
the confidence of the calculated ratios. In normal cases the color on the MAD value will be green or orange (with
more aberrations), when the MAD value becomes red you may need to try a different normalization factor or
adjust the reference probes for a more optimized normalization.
Figure 16 Different results sheets can also be used to explore your results. The colored sheets contain all important
results and data.
The sheet called RESULT (2) basically contains the same results but now with the 95% confidence range followed
after the estimated ratio. This 95% confidence range is the same as discussed earlier at the results reports. The
sheets called Reference runs and Sample runs contain the filtered raw peak height or areas (this can be set at the
settings option, in the tab called data filtering). Since 2008 on default the Coffalyser uses peak height even though
peak areas are theoretically more correct. This is done so, because several program often calculate the areas
underneath the peak incorrect, especially when the peak has a large base. Peak areas are thus only recommended
to use when the peaks contain little or no shoulders and when background signals are low.
Reference results sheet
Another important sheet is the reference results sheet. During the normalization a single reference signal is
created for each probe from all your reference runs. Before actual normalization of the samples, each separate
reference runs is also normalized against this signal. This not only allows the program to calculate the
reproducibility of each probe but can also inform you which reference runs failed. The results are displayed as
normal sample runs and each run should be completely blue in case your reference runs are performed on normal
DNA. The standard deviation over the reference runs should be low (it will be bold if the value is too high). If there
are reference runs that seem to contain aberrant probes, it is good to try to reanalyze your samples are deleting
the aberrant reference runs to see if results improve. This may also work when you set a median of your samples
runs as reference data (settings Tab, reference data). After the initial analysis where all your samples were set as
reference runs, you can explore your reference run results, recognize the samples that are most aberrant, then
reopen the coffalyser and delete these samples from the reference list. In a reanalysis the most normal looking
samples are now that as reference data which can improve your results.
Statistics sheet
The statistics sheet contains basic statistical information calculated over your reference runs. This sheet thus
doesnt give information about the reproducibility of your probes (unless all samples were normal runs), but rather
informs you on the probes that are changed most or least in the sample group used.
Predictions sheet
The predictions sheet can aid in results interpretation, although it should be used solely. The predictions are
furthermore only useful when at least 3 reference samples are used which were performed on normal human
DNA. The predictions are based on the same calculations as discussed earlier in the results report section. Each
prediction checks if the calculated ratio is more likely to be a gain (ratio 1,5) or loss (ratio 0.5). Signals that were
not found are automatically set as a homozygous deletion and ratios that were estimated higher than ratio 2.5 are
set as amplifications. Please note that most MLPA probes will not reach a ratio 1.5 in the case of a gain but rather
have a ratio between the 1.3-1.5. This makes the prediction less accurate and a gain may thus seem to be
ambiguous (or undetermined) while it is truly gained. True statistics are thus dependent on positive controls and
information about the MLPA probes signals in the case of a gain and also the reproducibility and spread of the
MLPA probes in the case of a gain (or loss).
A basic rule for MLPA probes ratios is that each aberrant probe signal should be confirmed by a second probe. This
can be a probe within the same sample results which has its target sequence close to that of the first probes or in a
second run or duplo.
1004.txt
1.01
1.03
1.03 1.09
1.04
1.08
1.14
1.04
1.08
1.45
1.02
1.05 1.00
1.02
1.01
1.01
1.03
0.99
1.45
1.05
1.06 1.03
0.99
1.07
1.04
1.02
1.02
1.34
0.95
0.99 0.99
0.93
1.05
0.96
1.02
1.00
1.42
0.89
0.94 0.93
0.88
0.92
0.93
0.94
0.90
1.41
0.99
1.04 1.03
1.03
1.06
1.00
1.00
1.00
1.50
1.05
0.97 1.04
1.04
0.99
0.95
1.02
0.99
1.41
0.47
0.95 0.98
0.95
0.94
1.04
1.04
0.94
1.45
0.51
1.03 1.00
1.04
1.03
0.99
1.00
1.00
1.45
0.44
0.89 0.90
0.86
0.92
1.06
0.97
0.91
0.97
0.99
1.12 1.09
1.03
1.06
1.06
1.01
1.05
0.97
1.03
1.04 1.08
1.03
1.04
1.08
1.05
1.06
0.90
0.93
0.99 1.01
0.94
1.04
1.04
1.02
1.00
0.93
0.98
1.04 1.09
1.01
1.03
0.99
0.96
1.06
0.96
1.06
0.99 1.06
1.01
1.05
1.03
0.99
0.99
1.00
1.00
1.06 1.05
1.02
1.03
1.04
0.96
0.98
0.96
0.98
0.99 1.02
0.96
1.06
0.99
1.02
0.99
0.96
0.99
0.99 1.07
0.95
0.90
0.96
0.93
1.01
1.04
1.11
1.07 1.06
1.05
1.00
1.02
0.99
1.05
0.97
1.05
0.96 0.94
0.89
0.95
0.86
0.95
0.90
0.96
0.96
1.00 0.98
0.97
0.98
1.00
0.97
1.00
0.96
1.04
1.02 1.12
1.03
1.08
1.07
1.01
1.03
0.98
0.97
0.99 0.99
1.01
0.98
0.98
1.02
0.99
1.00
1.02
1.01 1.03
0.97
1.02
1.00
1.03
1.03
1.01
0.96
0.00 0.51
0.97
0.00
1.00
0.97
0.97
1.01
1.11
0.00 0.52
1.01
0.00
1.06
1.01
1.02
1.00
1.00
0.00 0.49
0.96
0.00
1.00
1.01
1.00
0.98
1.11
1.03 0.53
1.03
0.00
0.00
1.01
1.06
1.01
0.92
0.98 0.50
0.85
0.00
0.00
0.95
0.93
1.06
0.96
0.97 0.47
0.91
0.00
0.00
0.90
0.97
0.98
0.97
1.06 0.97
1.00
1.04
1.04
1.03
1.02
1.03
1.02
1.06 1.04
1.00
1.06
1.01
1.01
1.03
0.98
1.02
1.07 1.00
1.03
1.06
1.03
1.04
1.07
0.99
1.00
1.00 0.96
1.00
0.97
0.97
1.00
0.98
1.00
1.05
1.01 1.01
1.02
1.03
1.01
1.00
1.03
1.03
1.03
1.08 0.90
0.97
1.03
0.95
0.99
1.04
1.02
1.05
1.00 1.03
1.06
1.01
0.98
0.99
1.03
1.04
1.06
1.02 1.08
1.04
1.00
1.03
1.00
1.05
0.95
1.03
0.98 0.97
0.98
0.99
0.99
0.98
0.98
0.99
1.04
1.01 0.96
0.95
0.97
0.95
0.99
1.04
0.98
0.92
1.05 0.97
0.99
0.99
1.02
0.99
1.00
1.03
0.90
0.92 0.96
1.02
1.01
0.93
0.95
0.94
1.03
1.06
0.88 0.97
0.95
1.00
0.92
0.94
0.48
0.99
0.95
1.01 1.02
0.98
0.98
1.01
1.02
0.46
0.99
0.94
0.89 0.95
0.93
0.93
0.82
0.96
0.44
Figure 17 Heatmap image of some samples with the P034 MLPA mix.