Is sequence upstream of P5/P7 okay?

[uberfinch]

Dear Sequencing gurus:


I would like to prepare a sequencing library that includes an upstream sequence (for the purposes of performing template suppression PCR on the library). Here is the structure of the envisioned library:


TS Sequence - P5 TruSeq - LIBRARY SEQUENCE - P7 TruSeq - TS Sequence

The TS sequence would ideally be 27 bases long.

Do you think that would interfere with cluster generation? Anyone have experience putting sequence upstream of P5/P7? I can always cleave it off with a restriction enzyme, but I'd like to avoid that will reduce library complexity.

Thanks in advance for your comments!

[kwaraska]

Since the P5 and P7 sites are what bind to the flow cell, I would assume that would interfere with binding. The P5/P7 sites bind to the oligos on the flow cell and they likely would not bind to it with a "tail"

[HeinKey]

27 bases might be too long. The "stuffer" between P5 and flow cell surface is probably shorter.
I would give it a try on a MiSeq nano run which is fairly cheap to test it. If it works it should be possible on Hiseq flow cells as well.