For QuantSeq REV, it is essential to use the Custom Sequencing Primer (CSP; (provided with the QuantSeq kit) for Read 1 sequencingPossible reasons can include:
Not using the CSP Version 5 for read 1 sequencing. This will result in a poly(T) stretch at the beginning of read 1, leading to reduced read quality and run output. Make sure that your sequencing facility is aware of
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these guidelines for CSP useage and that you supply them with the CSP Version 5 tube along with your samples or pool for sequencing: What is the CSP and how do I use it?
Mishybrization of the oligo(dT) primer during first strand synthesis steps of the library preparation protocol. If the temperature of the library prep is not maintained at 42 degrees Celcius, the oligo(dT) primer may mishybridize. This could also result in a minor fraction of library inserts being primed further into the poly(A) tail of the transcript. This can result in reads with longer poly(T) stretches at the start of read 1, despite the use of the CSP Version 5. Check our handling tips video for QuantSeq First Strand Synthesis to help avoid mishybridization in your library prep.