What is the difference between QuantSeq-Pool and QuantSeq FWD?

In both QuantSeq FWD and QuantSeq-Pool, library generation is initiated by oligo(dT) priming, where the primer contains a partial Illumina-compatible linker sequence.

However, in QuantSeq-Pool this primer also contains

• Unique Molecular Identifiers (UMIs), and

• an i1 sample-barcode

This early barcoding allows the samples to be combined by pooling after first strand synthesis. All subsequent steps of the protocol can thus be performed on pools containing up to 96 samples. Batch processing significantly shortens the overall library generation time and reduces technical variability between samples within one pool.

Additionally, the QuantSeq FWD and QuantSeq-Pool kits have different reagent compositions. As such, insert sizes are expected to differ. QuantSeq FWD generates library sizes averaging 335 – 456 bp, with mean insert sizes of 203 – 324 bp. QuantSeq-Pool generates library sizes longer than 600 bp, with inserts longer than 430 bp. Please note that the above references are based on Universal Human Reference RNA. Library shape and average insert size may vary, depending on a variety of factors e.g., sample type and sample quality.

For more information on differences in sample quality and input requirements, please check out the following FAQs:

Can I always replace standard QuantSeq with QuantSeq-Pool?

What factors should I consider when switching from QuantSeq FWD to QuantSeq-Pool?