Add a new instrument
Based on the imported datafile, Genotyper recognizes the used instrument (fluorescence reader) and will show this automatically in the instruments list.
If Genotyper cannot substract all instrument information from the input file, the instrument needs to be added manually by clicking on Add new instrument. This is necessary for array tape instruments, for example. Note that upon importing data from such a reader, you are forced to assign a reader that was created in the instruments module.
For every instrument, a user can change instrument specific parameters.
Tip:
Instruments for arraytape based PCR need to be added before importing data.
Instrument settings
General
Instrument name (on top of screen): This is the name that will be visualized by Genotyper for the user to easily know which instrument is used. The name can be edited to the user preferences.
Note that once a datafile is imported into Genotyper, the instrument name is set for this analysis. The edited instrument name will be applicable for the next analysis that is imported.Instrument ID: this is visualized in the top right corner of the plate view in an opened analysis. The instrument ID can’t be changed.
Instrument shortfall cutoff: all datapoints for which the passive reference fluorescence value is below this cutoff will be labelled as shortfall and will not receive a genotype call.
Instrument information: this free text field is optional and can be used for detailed information.
Minimal grid dimensions, see below.
Setting up minimal grid dimensions
For each instrument the minimal grid dimensions can be set. The algorithm will scale the data to this frame before analyzing the results.
If there is data outside of these dimensions, the visualization will be enlarged making sure history or outliers are taken into account.
This setting is useful for new assays without history, where in case of 1 cluster scoring can be difficult. By setting the minimal expected axes of the instrument, the orientation of the cluster can help making the decision.
The figure below shows an example without minimal grid axes (left) and with minimal grid settings (right):
By setting the minimal grid dimensions to the range that is known for the instrument, the cluster in this example will be located in the true area and scored XX.
Advanced
Warning:
These parameters influence the algorithm and thus the datapoint calling. Changing these parameters will render history irrelevant.
Note:
For Kraken, the advanced instrument settings are automatically import from Kraken and updated in Genotyper.
Deconvolution parameters
The deconvolution parameters adjust the influence of one fluorescence channel on the other. If clusters appear skewed or elongated along the diagonal (X ↔ Y contamination), increase deconvolutionX and deconvolutionY slightly.
For example, increasing the deconvolution Y, tilts the clusters towards the Y-axis:
Background correction
Removes unwanted baseline fluorescence from an experiment to improve data accuracy.
Normalization
Scales the fluorescence values to a standardized range.
Supported instruments
Don't see your instrument here? Contact [email protected] with some files to see if we can integrate your new hardware.
The following instruments are supported in Genotyper:
Bio-Rad
CFX family
BMG
Pherastar
LGC
Nexar, Araya
Intelliqube
Standard Biotools
Biomark (Fluidigm)
Thermo Fisher Scientific
QuantStudio
12K
5 (Dx)
6 (Pro)
7 (Pro)
ViiA7