Smart-3SEQ from scraped FFPE slides
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Grace Peters-Schulze
Oct 6, 2020, 7:01:28 PM10/6/20
to Smart-3SEQ
Hi Joe,
My name is Grace, and I'm Brooke Howitt's tech. I've been trying to get good bioanalyzer results for library prep from scraped slides. During my initial test runs, I got large adapter dimer peaks and a few smaller peaks around 200-350bp. Recently I tried a magnetic bead cleanup both pre and post-amplification. The most recent bioanalyzer graph doesn't show huge adapter dimer peaks and shows what I think is a better range of fragments, however concentration is still pretty low. Could you let me know what you think of this modification and if you feel the attached bioanalyzer graphs look good enough? I am thinking of increasing amplification from 16 to 20 cycles to increase concentration.
Thanks!!
Grace
Joe Foley
Oct 6, 2020, 8:37:58 PM10/6/20
to smart...@googlegroups.com
In the earliest stages of developing the protocol, we did an extra
cleanup before PCR, but we found it added too much variability.
Taking it out simplified the protocol while making the results more
consistent, but still good.
The region table says about 3.2 nM for sample "A+" (though it goes all the way up to 9000 bp and you might want to cut it off around 700 instead), which is indeed a little low, but it's more than enough for a sequencing run even if this is the only sample in the batch. Sample "C+" has some mild overamplification (slow-migrating fragments) so if anything I would reduce the PCR by one cycle from there.
There's basically no adapter dimer in sample "C+" but the amount in "A+" isn't terrible either. If you're still tweaking things and you were previously using the 0.7X cleanup, you could try switching to the 0.6X and see what you get, but then the yield might really become a problem. I would probably just leave it how it is, or use more input material if you have it.
The region table says about 3.2 nM for sample "A+" (though it goes all the way up to 9000 bp and you might want to cut it off around 700 instead), which is indeed a little low, but it's more than enough for a sequencing run even if this is the only sample in the batch. Sample "C+" has some mild overamplification (slow-migrating fragments) so if anything I would reduce the PCR by one cycle from there.
There's basically no adapter dimer in sample "C+" but the amount in "A+" isn't terrible either. If you're still tweaking things and you were previously using the 0.7X cleanup, you could try switching to the 0.6X and see what you get, but then the yield might really become a problem. I would probably just leave it how it is, or use more input material if you have it.
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