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Old 01-11-2016, 08:48 AM   #13
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Location: Oklahoma

Join Date: Sep 2009
Posts: 411

A couple more runs that don't meet spec.

Here's the first. It's two pools of NextFlex PCR-free on respective halves. Loaded at 150pM.
Q30 tanks towards the end:

We have a look at the %base and say "Ah Ha, sneaky adapter dimers slipping in!". Granted one should be able to clearly see adapter dimer traces on a Tapestation or Bioanalyzer but with PCR-free adapters things don't migrate properly and sizes are all off (we have tried denaturing and running it on a RNA tape, with some success).

Apparently the ExAmp and ordered flowcells just go bonkers with short libraries and that's what's killing the end of our reads, right?

So we take the two pools, give them a 0.8x bead cleanup (as opposed to our typical 0.9x) and load them back up again. We load at 100pM this time to really knock down any possibility of polyclonal wells.

The %base plot shows no more adapter dimers:

And read 1 Q30 looks pretty good relative to what you can expect on a 3k, but read 2 Q30 is an almost straight slope down:

I'm given to understand there's yet another lab out there that's having troubles with a 3k/4k. They've decided to not even bother going out to 150bp and just truncate the runs to 120bp. If that's the way this ends up going here I better not have to pay for those extra cycles.
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