# Assay

# Create a Assay Definition File (ADF)

The Assay Definition File (ADF) contains all required information which are needed to evaluate and analyse a data set with the appropriate assay.

• To add a new ADF click on the Create Assay Definition File.
• Choose the appropriate Result Type from the drop-down list.

Result type	File type
qPCR RDML	qPCR data in RDML data standard (.rdml)
qPCR RDML with melting temperature	Zip file containing the qPCR data in RDML data standard and melting temperature data in xml format (.zip)
Non-qPCR	All Non-qPCR data ()

• Fill the form and upload the ADF.
• The ADF uses a .xlsx format with the following parameters:

# for qPCR assay

qPCR parameter	Describtion	example/values	required
fluor	Fluorophore label for TaqMan probe or SYBR-Green/EvaGreen dyes	Cy5, FAM, or EvaGreen	yes
target	qPCR target name. Needs to be identical to the target name in the qPCR software.	18S	yes
pathogen	Species/Pathogen name, needed for generating reports	P. falciparum	yes
posctrl	Upper limit of acceptable Cq value for positive control	e.g. 25.0 (specific for each target)	if left empty, then the positive control will not be included into QC of qPCR run
negctrl	Acceptable Cq value for negative control	e.g. null (no amplification accepted)	if left empty, then the negative control will not be included into QC of qPCR run
ntc	Acceptable Cq value for NTC (non template control)	e.g. null (no amplification accepted)	if left empty, then the NTC will not be included into QC of qPCR run
quant	Quantification	e.g. yes (target is quantified)	no
threshold	Fluorescence threshold for Cq determination	e.g. 200	no
cutoff	Cq cut-off required for qualitative interpretation of qPCR result	e.g. 45.0	yes
minvalues	minimum number of replicates with same qualitative outcome required for each sample and target	e.g. 3 in case of triplicates	yes
cuttoffstdev	minimum number of replicates with same qualitative outcome required for each sample and target	0.1	yes
slope	slope for quantification based on linear regression	-0.251	yes, if quantification is desired
intercept	y-axis intercept for quantification based on linear regression	-0.251	yes, if quantification is desired
lod	target-specific limit of detection	0.05	no
qpcreff	amplification efficiency of a qPCR reaction based on the slope of the standard curve	0.99	no
reactionvol	total reaction volume in uL	20	no
samplevol	total reaction volume in uL	5	no
melt_min	lower limit of melting temparature range for dye-based qPCR	72.9	yes, if melting temperature needs to be considered
melt_max	uper limit of melting temparature range for dye-based qPCR	73.4	yes, if melting temperature needs to be considered

# for non-qPCR assay

Assay parameter	Describtion	example/values	required
target	Abbreviation of assay target	HIV test	yes
labels	possible assay outcomes for discrete* parameters	positive, negative, invalid	yes

(*) if the data is continous, leave empty

# Create an assay

The assay combines the Assay Definition File with additional relevant information from the Virtual Freezer and is through the Experiment linked to the Samples and qPCR data.

• Click Create Assay button.
• Enter the required (Name) and optional (Instrument and SOP) information.
• Select the appropriate Assay Definition File.
• Save the assay by clicking on the Create Assay button.