Small RNA library with 200bp extra peak

hoytpr · 02-12-2018, 03:18 PM

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

sbarberan · 02-12-2018, 04:05 PM

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

hoytpr · 02-12-2018, 04:47 PM

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.

sbarberan · 02-12-2018, 05:54 PM

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.

hoytpr · 02-13-2018, 08:53 AM

Quote:

Originally Posted by sbarberan

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?

sbarberan · 02-13-2018, 11:22 AM

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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02-12-2018, 03:18 PM	#1
hoytpr Member Location: Stillwater Join Date: Dec 2009 Posts: 62	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

02-12-2018, 04:47 PM	#3
hoytpr Member Location: Stillwater Join Date: Dec 2009 Posts: 62	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 04:51 PM.

02-12-2018, 04:05 PM	#2
sbarberan Member Location: Santa Cruz, CA Join Date: Feb 2017 Posts: 17	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

02-12-2018, 05:54 PM	#4
sbarberan Member Location: Santa Cruz, CA Join Date: Feb 2017 Posts: 17	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.

02-13-2018, 11:22 AM	#6
sbarberan Member Location: Santa Cruz, CA Join Date: Feb 2017 Posts: 17	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.