DNA Input Recommendations
The protocol supports 1−100 ng (where 1 ng is equivalent to ~300 genome copies) of human gDNA from high-quality sample or FFPE tissue. Recommended input is 10 ng high-quality DNA. 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 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-based 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.
Limited Samples
Degraded samples with average fragment sizes that are shorter than amplicon sizes can still yield Amplicon for Illumina Focus Panel libraries.
RNA Input Recommendations
The AmpliSeq Focus Panel RNA protocol reverse-transcribes RNA into cDNA. Each reverse transcription reaction requires 1–100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA. Before starting the protocol, quantify and dilute input RNA to the desired concentration.
- Increasing the amount of input RNA within this range typically results in higher-quality libraries, especially when RNA quality is unknown.
- Do not exceed the maximum supported amount of input RNA.
- Use 1 ng total RNA per pool only with high-quality, well-quantified samples.
- Isolate total RNA using a standard nucleic acid purification kit.
- Quantify the starting RNA using a fluorescence-based quantification method, such as the Qubit RNA HS Assay Kit or QuantiT RiboGreen RNA Assay Kit. Do not use a UV-spectrometer-based method.
- Library yield can be lower for degraded library samples such as FFPE RNA.