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Amcb1 04-03-2017 10:29 AM

Unexpected 425bp band un Truseq stranded mRNA library prep
3 Attachment(s)
I joined the community earlier today and I have a problem, I have carried out a Truseq Stranded mRNA HT library prep. and have an unexpected band in some of the samples. I used half of the same kit a few weeks ago with no problems and good sequencing. In other similar preps with the HT and LT kit the odd band has not been present, I have previously, occasionally seen adapter dimers at around 120 bp, which is explainable. This band is around 425 bp and is not present in all of the preps and is not present in the mock (water only) control, which makes me think that it is not in the core reagents. It looks like an amplicon, as it has a very sharp peak on the Tape station, and does amplify when I diluted 1:1000 and carried out a normal PCR with KAPA primers for 10 cycles. I am attaching the Tape station results for all of the libraries. If anyone has seem something similar or has any ideas I would be very grateful. At present my strategy will be to try a couple of samples on a nano cartridge on a Miseq and see if any of the results leap out as adapters or something unexpected.
With hope

nucacidhunter 04-03-2017 05:58 PM

I think it is most likely amplicon contamination if you do not have a designated pre- and post-PCR lab. You can also pay attention to see if the contaminated libraries are from particular columns or rows of the plate.

I have seen small extra peak (like GR-11, GR-23) when the TapeStation shakes during loading but most of yours are large peaks and the same size.

Amcb1 04-04-2017 02:19 AM

Thank you nucacidhunter,

I guess you are probably correct, if so I will see this when I carryout the test sequencing on the Miseq. You are also correct re. the pre and post PCR area at present although I am in the process of resolving this.
Thank you very much for your help.
best wishes

GA-J 04-05-2017 07:12 PM

Actually I think it may come from 18s or 28s if your starting mass was too high.I saw some peaks from mRNA lib that started from poor quality of RNA. Not for sure, just a guess.

Amcb1 04-06-2017 02:19 AM

Thank you GA-J,
It's possible as most of the extra bands are present in samples that had the higher concentration of starting material, however it should have been normalized to 500ng per sample after the initial Qubit. Also it does not entirely correlate with starting concentrations or RIN scores. I should find out after I check a couple of the samples on a nano cartridge. I will post what I find for information to everyone in case this is helpful.
Many thanks to you and all who had read my post and thought about it.

RickC7 06-07-2017 11:16 AM

Did you ever figure out what the peak was? I have a similar issue...

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