When quantifying sequencing libraries, take into account all products, off-target and on-target, that may cluster on the flow cell. We recommend using Bioanalyzer or Tapestation/Fragment Analyzer to quantify individual tube libraries.
Set the size range to 100 – 700 bp to account for off-target, e.g., primer dimers, and on-target amplicons. Normalize the concentration of each tube library to 5 nM using the Tapestri Library Quantification Tool. Pool the equimolar libraries and re-quantify with Qubit to confirm a final concentration of 0.9 – 1.3 ng/µL (~ 5 nM).
Alternatively, the library may be quantified using quantitative PCR (qPCR) using the KAPA Library Quantification Kits.
The Myeloid-panel libraries are prone to generate large-size off-target products due to extended annealing/extension times during the thermal cycling protocol, e.g., concatemers. They need to be taken into consideration when quantifying the libraries.
- Set the size range from 100 – 700 bp to quantify all products that may cluster on the flow cell.
- Set the size range from 700 – max, excluding the marker, to quantify all products that are unlikely to efficiently cluster on the flow cell but will contribute to the overall concentration when re-quantifying the pooled library via Qubit.
- Normalize the tube libraries to 5 nM based on the first measurement (100 – 700 bp) and pool the equimolar libraries.
- Re-quantify the pooled library via Qubit. The final concentration will likely be above 1.3 ng/µL (~5 nM).
- Alternatively, the library may be quantified using quantitative PCR (qPCR) using the KAPA Library Quantification Kits.