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**HCS-Tools for KNIME** version **4.0.0.v202007151505** by **Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)**

Groups of data (e.g. wells of a plates) are normalized relative to two subsets (e.g. some wells within that plate, e.g. some negative control wells and some positive controls wells). For each group the mean (or median) is calculated for the two selected subsets. Based on these estimates, all data points of each group are normalized by applying this formula:

x.npi = (mean(x[subset{positive}]) - x) / (mean(x[subset{positive}]) - mean(x[subset{negative}])) * 100

or

x.npi = (median(x[subset{positive}]) - x) / (median(x[subset{positive}]) - median(x[subset{negative}]))
* 100

Therfore x.npi values resemble the "percentage of inhibition", where the positive subset is defined as 0% and the negative subset as 100%. By swapping the two subsets the definition will be "percentage of activation" and negatives will be set to 0% and positives to 100%.

Literature: Malo et al., Nat. Biotechnol. 24, 167-179 (2006)

- Group data by
- Select the column to define the groups (e.g. "barcode" for plate-wise normalization). Select >none< for no grouping (e.g. normalization of the whole screen based on all negative controls available).
- Normalize
- Select the columns with data to be normalized.
- Column with reference label
- Select the column which contains the label defining the reference subset (e.g. treatment)
- Subset / Positive Control
- Select a subset (representing maximum activation) that is used to calculate mean (median) for each group
- Subset / Negative Control
- Select a subset (representing maximum inhibition) that is used to calculate mean (median) for each group
- Replace existing values
- If checked the raw values will be replaced by the normalized values. Otherwise columns with the normalized values will be appended - ".npi"
- Use robust statistics
- Median will be calculated instead of mean.
- Column suffix
- The default column suffix ".npi" can be changed
- Load a limited number of columns
- If big data tables create memory problems, the number of columns read in memory (subset data) can be restricted. Then, statistic calculation will be done sequential for each set with the given number of columns.

- Column Filter (15 %) Streamable
- Row Filter (9 %) Streamable
- Plate Heatmap Viewer (8 %)
- Joiner (8 %)
- Normalize Plates (POC) (7 %)
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~~CSV Writer~~(11 %) StreamableDeprecated- Row Filter (11 %) Streamable
- GroupBy (8 %)
- Column Filter (8 %) Streamable
- Normalize Plates (POC) (7 %)
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- 03_Normalization_of_screening_data (KNIME Hub)

To use this node in KNIME, install KNIME HCS Tools from the following update site:

KNIME 4.3

A zipped version of the software site can be downloaded here.

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