This kit is based on absolute quantification of your NGS libraries using Illumina adaptor primers and qPCR. The amplifiable fraction of the library will get amplified, and the quantity will be estimated using a standard curve. The goal of the analysis is to find the optimum concentration for the NGS library, since over- or underestimation of the concentration may compromise the sequencing capacity.
- Underestimation of the concentration → Overloading of the flow cell → Poor cluster resolution
- Overestimation of the concentration → Insufficient template amount → Low cluster density
Six DNA standards of known concentrations are part of the kit for generation of a robust standard curve. They will generate PCR products of 415 bp. After diluting your NGS library of unknown concentration 10 000 times and performing qPCR analysis, the library concentration is assessed. PCR based library preps yield mostly concentrations of 1-50 nM.
• NGS library assay primers (V = 250 μl primer mix, c = 10 μM per primer, Blue cap)
• TATAA SYBR GrandMaster Mix LowROX (V = 2 · 1250 µl, c = 2x)
• NGS library standards: 10-fold diluted Standard 1-6, dsDNA in TE buffer (V = 40 µl per standard)