What are the differences between QuantSeq REV and FWD?

With QuantSeq Reverse (REV, Cat. No. 225), the Read 1 adapter sequence is located on the 5' end of the oligo(dT) primer and the Read 2 adapter sequence is located on the 5' end of the random primers used during second strand synthesis. This generates libraries that have a reverse orientation compared to those generated by the QuantSeq FWD kit.

Open

Figure | Schematic overview of the QuantSeq REV V2 library preparation workflow (Cat. No. 225). Sequencing
read orientation for QuantSeq REV is depicted, with Read 1 (green) and Read 2 (blue). Read 1 reflects the cDNA sequence. QuantSeq REV is suitable for paired-end sequencing, and Read 2 reflects the mRNA sequence. A Custom Sequencing Primer (CSP Version 5, included in the kit) is required for Read 1.

A Custom Sequencing Primer (CSP, included in the kit) is required for sequencing QuantSeq REV libraries, to avoid sequencing the poly(T) stretch at the start of Read 1. This enables Read 1 to begin at the exact 3' end of the transcript.

Using Paired-End sequencing is advantageous to identify 3' UTR isoforms.

The use of QuantSeq REV is therefore ideal for applications including alternative polyadenylation site identification and alternative 3' UTR expression studies.