Can I always replace QuantSeq FWD with QuantSeq-Pool?

No, QuantSeq-Pool uses early pooling and thus equal read distribution in sequencing experiments requires normalization of the input RNA amount prior to starting library generation. This can be achieved best with high quality RNA (RIN > 6) with at least 10 ng per sample.

The RNA input should also be homogenous in terms of integrity, purity, and quantity across all samples.

Pooling non homogeneous samples may result in unequal read share and hence, suboptimal read quality for some samples.

For further information please consult the Appendix information in the QuantSeq-Pool Sample-Barcoded 3' mRNA-Seq User Guide and the following online FAQ: What factors should I consider when switching from QuantSeq FWD to QuantSeq-Pool?