(2x) Final Bead Clean-Up

[cement_head]

Hello,

I have a general question regarding NGS library prep protocols; many of the protocols suggest using TWO rounds of bead clean-up at the end of the protocol. I am curious as to why TWO (2) rounds of clean-up. Shouldn't one "proper" iteration (round) be enough? Or are the companies worried about primer dimers so much that they are trying to CTAs and suggest two rounds?

Thanks-in-advance...

[jdk787]

I have seen many PCR Free protocols recommend two cleanups to be sure to get rid of any unincorporated adapters which are hard to detect and known to cause index hopping.

This could also be needed after PCR for protocols that use PCR primers that include index sequences.

[luc]

Yep, bead cleanups will never be perfect. You can only enrich for molecules of the desired size range.
Many protocols suggest two rounds of bead cleanups likey to safeguard for less than optimal experiments that created too many adapter dimers.