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Old 02-12-2018, 02:18 PM   #1
hoytpr
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Default Small RNA library with 200bp extra peak

I just looked at our new small RNA library submitted by a respected lab on the Bioanalyzer. The small RNA library should be about 178, and it's there, but most of the samples have a broader but distinct peak at 200bp. I've posted a link to an example below. Any ideas what this could be and even more, how to eliminate it?

Thanks.
Pete
https://drive.google.com/open?id=1Rl...VedTDyXROD1mKt
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Old 02-12-2018, 03:05 PM   #2
sbarberan
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That to me looks like over-amplification, we have seen 'bulged' products that run at 200-250bp. See page 9 of this protocol from the Mello lab (http://www.lsi.umich.edu/files/SmallRNACloning.pdf)

What kit was used to prepare the libraries? 178 is big for miRNAs unless you are using QIAseq.

Cheers,
sergio
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Old 02-12-2018, 03:47 PM   #3
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You're right, that does resemble over amplification. I was going to run the samples on a denaturing gel but that protocol says not to. Can it be fixed? I think the protocol was mostly custom. I'm not someone who makes small RNA libraries yet, so I can't really comment on what was custom and what may have come from a kit.

Last edited by hoytpr; 02-12-2018 at 03:51 PM.
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Old 02-12-2018, 04:54 PM   #4
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Bulged products are not a problem for sequencing, you only see them with gels or bioanalyzer. For sequencing you denature the libraries anyways. So there is nothing to fix, just that they probably amplified the libraries for too many cycles.
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Old 02-13-2018, 07:53 AM   #5
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Quote:
Originally Posted by sbarberan View Post
Bulged products are not a problem for sequencing, you only see them with gels or bioanalyzer. For sequencing you denature the libraries anyways. So there is nothing to fix, just that they probably amplified the libraries for too many cycles.
So, you'd have to quantify by QPCR then, as fluorescence or Bioanalyzers won't tell you the number of correctly formatted sequences with adapters?
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Old 02-13-2018, 10:22 AM   #6
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You are probably right that qPCR will be the only way to quantify with 100% accuracy, but generally we do not use qPCR and even with libraries with bulged products we manage to quantify only with Qubit and Bioanalyzer and get great pooling.

Alternatively you can also try to denature the sample before loading into the Bioanalyzer.
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