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miseq_user 04-06-2020 08:22 AM

Library with strong secondary structures
What is known about the strand displacement activity of the DNA polymerase Illumina uses for bridge amplification? I have a library with strong secondary structures and I am worried that clusters do not form well due to the secondary structures. Does anyone have any tips to improve cluster formation for such a library?

GenoMax 04-06-2020 11:50 AM

Anecdotally we find MiSeq to be the best sequencer for odd samples. You best bet is to sequence this library there. You can always a run a nano flowcell at a much reduced cost to test before you run full library.

misterc 04-08-2020 02:36 PM

You can also try a 95C denaturation (instead of the usual NaOH denaturation). 1 minute at that temperature (in RSB or EB should be fine), just make sure you have a decent volume there so you don't evaporate everything. TruSeq Custom Amplicon product does this method to make the libraries nice and single stranded for cluster generation.

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