When to quantify libraries and method of quantification for Illumina DNA PCR Free libraries

Can libraries still be manually quantified and normalized if over 300 ng is used (saturating input)? Yes, libraries can be quantified when input DNA is over the saturation point. For pooling, pool 9 µl or more per sample to mitigate pipetting variability.

If the BLT-PF is self-normalizing at saturating conditions, why is a quantification after pooling still recommended? This is because user to user and experiment to experiment variation is higher than sample to sample variation. Quantification is also required to calculate how to dilute libraries to the starting loading concentration.

Can qPCR quantification be used for Hybex workflow? Yes, KAPA qPCR is suitable for all input amounts, but is required for low DNA input (<300 ng).

Should Qubit or qPCR quantification be used for saliva/blood/dried blood spots?

• Either qPCR or single-stranded (ssDNA) Qubit quantification can be performed.

Is it possible to omit the final pool quantification?

No, libraries must be quantified prior to sequencing.

For any feedback or questions regarding this article (Illumina Knowledge Article #3476), contact Illumina Technical Support [e-mail].