BatchKi Reference Manual

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Plate Reader Output

Plate reader data can be input into the program in one of several different formats:

1. SoftMax ``plate'' format (Molecular Devices, Inc., moldev.com)
2. SoftMax ``column'' format (Molecular Devices, Inc., moldev.com)
3. Bio-Tek format (Bio-Tek Instruments, Inc., biotek.com)
4. Bio-Rad format (Bio-Rad Laboratories, Inc., bio-rad.com)
5. Universal tabular format
6. Two-column format

The details of each data format are explained below. The raw or modified plate reader data are contained within the <Data...> element of the BatchKi input file.

The <Data...> element

The general form of this element in the BatchKi input file is shown below.

  <Data type="[progress|rate]"
        format="[ThermoMaxColumn|ThermoMaxPlate|Universal|...]"
        header="[0|1|2|3|...]"
        external="no"
        background="...">
   ...
   ...
  </Data>

The type attribute

The type attribute can have one of two values:

• type = "progress"
• type = "rate"

Reaction progress as primary data

When type is set to progress, it is assumed that the primary experimental data produced by the plate reader are reaction progress curves. In these experimental situations, the enzyme assay is continuous and consists of readings of several primary data points (such as absorbance or fluorescence) over the course of time for each experiment.

The exact format of the reaction progress data is determined by the format attribute (see below). The type = "progress" is the default value for the program and thus it can be omitted from the data file.

Initial velocities as primary data

When type is set to rate, it is assumed that the primary experimental data produced by the plate reader are initial reaction velocities, or some type of end-point measurement originating in a cell-based assay.

End-point assays often involve (a) an equilibration of the enzyme with the inhibitor, (b) reaction with the substrate for a fixed period of time, and (c) end-point readout of a physical quantity such as radioactivity (cpm). Under these circumstances, it is important that the reaction velocity stays approximately constant during the reaction period. In other words, it is important to keep the final substrate conversion very low, typically within a few percentage points.

When the reaction velocities or other end-point data are used, the contents of the <Data> section must be arranged in a tab-delimited text format, such that the proper number of rows and columns corresponds to the number of rows and columns on the given plate.

The header attribute

The header attribute must be a whole number (1, 2, 3, etc.) or zero:

• header = "0"
• header = "1"
• header = "3"
• header = "8"
• etc.

The program will interpret each value of the attribute as is described below for each particular format (ThermoMaxColumn, ThermoMaxPlate, Universal, or TwoColumn).

The external attribute

As was explained above in section 5.4.2, the external attribute must contain one possible value:

• external = "no"

This attribute determines whether the concentration information is embedded in the BatchKi XML data file, or whether the concentration information is contained in a separate text file.

The format attribute

The format attribute can have one of several values, corresponding to different plate-reader formats:

• format = "ThermoMaxColumn"
• format = "ThermoMaxPlate"
• format = "Bio-Tek"
• format = "Bio-Rad"
• format = "Universal"
• format = "TwoColumn"

SoftMax ``Column'' format (ThermoMaxColumn)

The SoftMax format is a tabular format with the following characteristics:

• The raw data block contains 98 columns of ASCII text.
• All columns are delimited by the `tab' character.
• The first column is time in the format `minutes:seconds'.
• The second column is temperature (ignored by the program).
• Columns 3 through 98 are readings of experimental signal.
• This format is identified in the BatchKi input by the keyword

  <Data format="ThermoMaxColumn" ...>.

• The raw data be preceded by zero or more header lines (for example, a header line containing text such as ``A1 A2 ... H12''); the header lines are ignored by the program, but if they are present, they must be identified by the keyword header followed by the appropriate number of lines:

  <Data ... header="[0|1|2|3|...]" ...> .

Example - SoftMax ``Column'' format

An example of a SoftMax ``Column'' format, with two (manually added) header lines, is shown below:

TIME   TEMP   A1        A2        ...   H11       H12
----   ----   -------   -------         -------   -------
0:00   23.4   175.947   403.869   ...   305.405   313.048
0:29   23.4   175.944   403.533   ...   356.039   362.015
0:58   23.4   178.393   403.878   ...   403.908   408.971
1:27   23.4   178.520   407.875   ...   449.087   454.643
1:56   23.4   179.504   404.839   ...   495.273   499.109
2:25   23.4   177.524   403.569   ...   541.067   543.522
2:54   23.4   176.481   402.379   ...   583.401   587.465
3:23   23.5   178.185   403.747   ...   628.262   629.192
3:52   23.5   176.971   403.225   ...   671.813   672.791
4:21   23.5   176.727   403.164   ...   712.987   714.830
4:50   23.5   177.453   404.628   ...   756.297   757.106

The SoftMax ``Column'' format is automatically produced by the Molecular Devices software.

SoftMax ``Plate'' format (ThermoMaxPlate)

The SoftMax format is a tabular format with the following characteristics:

• The raw data file contains 14 columns of ASCII text.
• All columns are delimited by the `tab' character.
• Data blocks are repeated in blocks of 13 rows, each block corresponding to a single time-point.
• The first column is time in the format `minutes:seconds'.
• The second column is temperature (ignored by the program).
• Columns 3 through 14 are readings of experimental signal.
• This format is identified in the BatchKi input by the keyword

  <Data format="ThermoMaxPlate" ...> .

• The raw data be preceded by zero or more header lines (for example, a header line containing text such as ``A1 A2 ... H12''); the header lines are ignored by the program, but if they are present, they must be identified by the keyword header followed by the appropriate number of lines:

  <Data ... header="[0|1|2|3|...]" ...>.

Example - SoftMax ``Plate'' format

0:00   23.4   175.947   403.869   ...   196.843   312.949
              180.296   238.717   ...   227.373   318.621
              180.194   192.757   ...   271.300   321.673
              180.147   181.931   ...   310.313   323.704
              185.928   179.083   ...   314.858   325.602
              197.600   178.256   ...   313.200   319.688
              215.462   179.196   ...   317.814   311.291
              233.514   189.411   ...   305.405   313.048

0:29   23.4   175.944   403.533   ...   202.405   363.904
              179.601   238.447   ...   243.698   368.824
              179.916   191.289   ...   305.696   373.639
              181.889   179.974   ...   357.386   375.547
              193.857   178.331   ...   366.236   377.531
              221.750   176.657   ...   364.033   370.917
              256.139   179.792   ...   370.059   360.878
              283.235   190.257   ...   356.039   362.015

...

The SoftMax ``Plate'' format is automatically produced by the Molecular Devices software.

Bio-Tek format (Bio-Tek)

The Bio-Tek format is a semicolon-separated, tabular format with the following characteristics:

• Each time point is represented by a block of $n + 4$ lines of text, where $n$ is the number of rows on the plate.
• The first line in each block is empty.
• The second and fourth lines can be ignored as headers.
• The third line contains the reading of time in the format `hours:minutes:seconds'.
• The fifth and subsequent lines represent the signal (e.g., absorbance) for one row on the given plate.
• Each data row begin with a row designation (e.g.k A, B, etc.).
• The row designation is followed by $m$ data items (e.g., absorbance readings), where $m$ is the number of columns on the plate.
• All data items are separated by semicolons.
• This format is identified in the PlateKi input by the keyword

  <Data format="Bio-Tek" ...> .

The Bio-Tek format is automatically produced by the KC4 software (Bio-Tek Instruments, Inc., biotek.com).

Example - Bio-Tek format

This fragment shows the first two time points (0 seconds and 21 seconds) of a Bio-Tek data file format.

[Plate: M#1 405 Corr.  #1]
Time=00:00:00
;1;2;3;...;10;11;12
A;-0.011;0.007;0.043;...;0.060;0.076;-0.007
B;-0.019;0.015;0.019;...;0.052;0.089;0.006
C;0.002;0.014;0.022;...;0.048;0.068;0.000
D;0.000;0.015;0.021;...;0.069;0.083;0.008
E;-0.003;0.014;0.032;...;0.074;0.080;0.010
F;0.008;0.017;0.014;...;0.067;0.089;-0.010
G;-0.009;-0.009;0.008;...;0.065;0.079;-0.008
H;0.013;0.002;0.007;...;0.069;0.082;0.001
                               
[Plate: M#1 405 Corr.  #2]
Time=00:00:21
;1;2;3;...;10;11;12
A;-0.014;-0.009;0.031;...;0.066;0.084;-0.007
B;-0.023;-0.005;0.013;...;0.057;0.097;0.004
C;-0.004;0.009;0.017;...;0.056;0.075;0.003
D;-0.006;0.010;0.017;...;0.076;0.093;0.009
E;-0.005;0.008;0.024;...;0.081;0.089;0.008
F;0.006;0.015;0.014;...;0.074;0.097;-0.009
G;-0.011;-0.013;0.007;...;0.072;0.081;-0.010
H;0.008;0.001;0.007;...;0.077;0.089;-0.002
                
...   ...   ...

Bio-Rad format (Bio-Rad)

This format is identified in the PlateKi input by the keyword

<Data format="Bio-Rad" ...> ;

The Bio-Rad format is automatically produced by the Microplate Manager software from Bio-Rad Laboratories, Inc. (bio-rad.com).

Example - Bio-Rad format

The fragment below shows the first two and the last two time points (columns) in a typical Bio-Rad data file:

16:33:22   16:33:29   ...   16:43:18   16:43:26
0.309      0.309      ...   0.369      0.371
0.309      0.309      ...   0.37       0.371
0.305      0.307      ...   0.364      0.365
...        ...        ...   ...        ...
...        ...        ...   ...        ...
...        ...        ...   ...        ...
0.315      0.321      ...   0.387      0.385
0.31       0.314      ...   0.383      0.381

Note that the spacing of time points in a Bio-Rad file is not necessarily uniform. In the example above, the interval between the first two time points is seven seconds, whereas the interval between the last two time points is eight seconds. BatchKi converts the raw data into a uniformly spaced time points, which is required for certain computational algorithms (e.g., exponential fit using the Legendre polynomial method [6]).

Universal format (Universal)

The universal tabular format is similar to the SoftMax format described above with the following exceptions:

• The temperature column is nor present.
• The first column contains time in seconds in decimal format (e.g., `297' instead of `4:57').
• The number of rows and columns in not limited to 8 and 12, respectively.
• The format is identified in the BatchKi input files as

  <Data format="Universal" ...>.

The universal tabular format can be produced by proprietary software tools and scripts from the SoftMax output files, or from any other primary data files.

Raw plate reader data in the Universal tabular format can contain zero or more header lines. If such lines are present, the flag header must be set as is shown above for the SoftMax format.

Example - Universal format

An example of the Universal data format with two header lines is shown below:

TIME   A1        A2        ...   H11       H12
----   -------   -------         -------   -------
   0   175.947   403.869   ...   305.405   313.048
  29   175.944   403.533   ...   356.039   362.015
  58   178.393   403.878   ...   403.908   408.971
  87   178.520   407.875   ...   449.087   454.643
 116   179.504   404.839   ...   495.273   499.109
 145   177.524   403.569   ...   541.067   543.522
 174   176.481   402.379   ...   583.401   587.465
 203   178.185   403.747   ...   628.262   629.192
 232   176.971   403.225   ...   671.813   672.791
 261   176.727   403.164   ...   712.987   714.830
 290   177.453   404.628   ...   756.297   757.106

Two-column format (TwoColumn)

The two-column format is most suitable for two types of experimental situations:

1. The time values are not the same for all wells on the plate.
2. The plate contains more than 256 wells (e.g., it is a 16 $\times$ 24 plate), which makes it difficult or impossible to manipulate in a tabular format by using conventional spreadsheet programs (e.g., Excel).

Raw data in the two-column format are presented as two tab-delimited columns of ASCII text. The first column contains time in seconds or minutes and the second column contains the corresponding readings of the experimental signal.

To increase readability of the raw data file, each two-column data set (a progress curves collected for a particular well) may be preceded by a header line with an arbitrary text.

Example - Two-column format with header lines

<Data format="TwoColumn" header="1"...>
ROW#1 COLUMN#1 WELL#1
0	0.075
11	0.074
22	0.076
33	0.077
...
ROW#1 COLUMN#2 WELL#2
0	0.103
11	0.103
22	0.104
33	0.104
...
...
ROW#16 COLUMN#24 WELL#384
0	0.081
11	0.086
22	0.091
33	0.096
</Data>

Example - Two-column format without header lines

<Data format="TwoColumn" header="0"...>
0	0.075
11	0.074
22	0.076
33	0.077
...
0	0.103
11	0.103
22	0.104
33	0.104
...
...
0	0.081
11	0.086
22	0.091
33	0.096
</Data>

The number of consecutive blocks of two-column tab delimited text, with each block holding raw data collected from a single well, must correspond to the number of rows and columns given by the attributes rows="..." and columns="..." specified in the <Plate...> element.

EXAMPLE

Let us assume that each progress curve contains 28 data points, and that each progress curve segment is preceded by a single header line. Furthermore let us assume that the given plate contains 16 rows and 24 columns, which means there are 16 $\times$ 24 = 384 wells. Thus, the <Data...> section of the BatchKi input file must contain exactly 384 $\times$ (28 + 1) = 11,136 lines of ASCII text.

The background attribute

This attribute of the <Data> element is optional. When present, the attribute defines the background (uncatalyzed) component for all reaction rates observed on the given plate.

The ability to define the background rate is important for example in the analysis of rotamase assays, or in the analysis or radioactive end-point assays of certain kinases. The exact interpretation of the numerical value given by background is explained in section 4.5.14 (see Algorithm 1).

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biokin.com/batchki/manual/reference/html/node27.html
Petr Kuzmic | Jul 12 2008