Use PreSeq® to determine library input
To determine DNA input quantity to use for VariantPlex® assays, calculate your DNA QC Score using Equation 1. Input your DNA QC Score into the calculator below to review application-specific input requirements particular to your sample.
Calculate ng input for different DNA/VariantPlex applications
NOTE: The DNA QC Score is calculated assuming you did not deviate from the PreSeq® DNA QC Assay protocol (using 5ng input).
In general, using more input material will result in better assay performance (up to the maximum).
|50 Unique mean
WARNING: We do not recommend using greater than input with our assays. Using more than input may produce unpredictable/low-quality results.
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How we calculate amplifiable genomes from reference DNA
The PreSeq® DNA QC assay evaluates the quantity of amplifiable DNA in a sample relative to a control template. In two separate reactions primers target a 100bp genomic region and synthetic template region. Comparison of the two quantification cycles (Cq) results in a DNA QC score, a ΔCq value (See Equation 1), providing a quantitative measure of DNA quality. This also permits estimating the concentration of the sequencable copies of genomic DNA present in the sample.
We define an amplifiable genome as one complete haploid genome of sufficient quality and fragment length to be detectable by PCR. Thus, for each amplifiable genome present in a sample, there is on average a single copy of each genomic locus that is available for library generation.
The number of amplifiable genomes is a function of input DNA mass and DNA QC score for a sample. The Synthetic Standard provided in the PreSeq® DNA QC Assay is a non-human control sequence of known concentration, permitting calculation of amplifiable genomes using Equation 2.