DNA Input Recommendations
The protocol supports 1-50 ng per pool (where 1 ng is equivalent to ~300 genome copies) of human gDNA from normal tissue or FFPE tissue. Recommended input is 10 ng high-quality DNA per pool. Before starting the protocol, quantify and dilute input DNA to the desired concentration.
- Increasing the amount of input DNA within this range typically results in higher library quality, especially when DNA quality is unknown.
- Do not exceed the maximum supported amount of input DNA.
- Use 1 ng gDNA per pool only with high-quality, well-quantified samples.
- Library yield can be lower for degraded library samples such as FFPE DNA. Inhibitors such as high melanin content can reduce the efficiency of target amplification.
Input DNA Quantification
Quantify the starting DNA using a fluorescence-based quantification method, such as a Qubit dsDNA HS Assay Kit or PicoGreen. Do not use a UV spectrometer method.
- Fluorescence-based methods employ a dye specific to double-stranded DNA (dsDNA) and specifically and accurately quantify dsDNA, even when many common contaminants are present.
- In contrast, UV spectrometer methods based on 260 OD readings can overestimate DNA concentrations. The overestimation is due to the presence of RNA and other contaminants common to gDNA preparations.
Degraded samples with average fragment sizes that are shorter than amplicon sizes can still yield Amplicon for Illumina BRCA Panel libraries.