Bioanalyzer: Multiple Peaks after library PCR

HarryHaller89 · 01-26-2014, 04:34 AM

Hello,

in some of my prepared sequencing libraries I observe multiple peaks in the bioanalyzer run after library pcr (see attached file). Many of the peaks have an average distance of arround 60 bp. Are these adaptor concatameres or something like this?

Is there any way to avoid these peaks?

I know the peak at 126 bp are adaptor dimers... But this is a different story.

Best Harry

NextGenSeq · 01-28-2014, 10:43 AM

That is the strangest library I've ever seen. What type of library is this?

HarryHaller89 · 01-28-2014, 10:52 AM

It's a ChIP-Seq library prepared with Illumina's TrueSeq ChIP Prep kit - prepared according to the protocol. I have it in both input samples and IP samples. However, from around 50 samples it happened only in 6.

My first idea was that it depends on the initial amount of DNA used for library preparation, but there is absolutely no correlation (it happend both for the maximal amount 10 ng and for a lower input amount 5-6 ng). The bioanalyzer traces are reproducible, excluding problems with the run. I personally can not believe that these are adaptor concatamers - since these peaks appear up to 1800 bp (would basically mean 30 adaptors ligated...).

pmiguel · 01-28-2014, 11:16 AM

Did you happen to use the spike-in standards for library construction? Those can give some strange patterns.

--
Phillip

HarryHaller89 · 01-28-2014, 11:28 AM

No, nothing was added during library preparation.

Samples were prepared in parallel from the same fragmented chromatin and only few of them showed this pattern. All other libraries look fine. It also does not depend on particular adaptors.

Could these peaks be the result of contaminations in the samples?

pmiguel · 01-28-2014, 11:42 AM

Quote:

Originally Posted by HarryHaller89

No, nothing was added during library preparation.

Samples were prepared in parallel from the same fragmented chromatin and only few of them showed this pattern. All other libraries look fine. It also does not depend on particular adaptors.

Could these peaks be the result of contaminations in the samples?

Sure. Was there any gel purification step before the ligation? Some of the samples might have been near to a molecular weight marker that contaminated the resulting sample.

--
Phillip

HarryHaller89 · 01-28-2014, 11:49 AM

No the gel purification was performed after the ligation step. The strange samples were not near the markers (50 bp ladder) on the gels.

barrmur · 01-28-2014, 12:46 PM

Silly question first. Have you run them on the chip a second time? It almost looks like bleed through from the ladder to be honest. Adapter concatamers is also possible but we have done a lot of chip preps and I have never seen this.

HarryHaller89 · 01-28-2014, 10:59 PM

Quote:

Originally Posted by barrmur

Silly question first. Have you run them on the chip a second time? It almost looks like bleed through from the ladder to be honest. Adapter concatamers is also possible but we have done a lot of chip preps and I have never seen this.

Yes, the pattern is absolutely reproducible and not dependent on a particular position on the chip or anything like this.

NextGenSeq · 01-31-2014, 08:33 AM

The 126 bp peak is an adapter dimer. The other peaks are likely concatamers of the adapter dimer.

I wouldn't bother sequencing this library. I would just remake it.

windnature03 · 06-21-2016, 12:50 AM

how about sequencing the sample??
Were there any strange result or it is just a problem on bioanalyzer??

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01-28-2014, 12:46 PM	#8
barrmur Member Location: Nottingham, England Join Date: Nov 2009 Posts: 26	Silly question first. Have you run them on the chip a second time? It almost looks like bleed through from the ladder to be honest. Adapter concatamers is also possible but we have done a lot of chip preps and I have never seen this. Last edited by barrmur; 01-28-2014 at 12:50 PM.

01-28-2014, 10:43 AM	#2
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	That is the strangest library I've ever seen. What type of library is this?

01-28-2014, 10:52 AM	#3
HarryHaller89 Junior Member Location: Germany Join Date: Jan 2014 Posts: 5	It's a ChIP-Seq library prepared with Illumina's TrueSeq ChIP Prep kit - prepared according to the protocol. I have it in both input samples and IP samples. However, from around 50 samples it happened only in 6. My first idea was that it depends on the initial amount of DNA used for library preparation, but there is absolutely no correlation (it happend both for the maximal amount 10 ng and for a lower input amount 5-6 ng). The bioanalyzer traces are reproducible, excluding problems with the run. I personally can not believe that these are adaptor concatamers - since these peaks appear up to 1800 bp (would basically mean 30 adaptors ligated...).

01-28-2014, 11:16 AM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Did you happen to use the spike-in standards for library construction? Those can give some strange patterns. -- Phillip

01-28-2014, 11:28 AM	#5
HarryHaller89 Junior Member Location: Germany Join Date: Jan 2014 Posts: 5	No, nothing was added during library preparation. Samples were prepared in parallel from the same fragmented chromatin and only few of them showed this pattern. All other libraries look fine. It also does not depend on particular adaptors. Could these peaks be the result of contaminations in the samples?

01-28-2014, 11:49 AM	#7
HarryHaller89 Junior Member Location: Germany Join Date: Jan 2014 Posts: 5	No the gel purification was performed after the ligation step. The strange samples were not near the markers (50 bp ladder) on the gels.

01-31-2014, 08:33 AM	#10
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	The 126 bp peak is an adapter dimer. The other peaks are likely concatamers of the adapter dimer. I wouldn't bother sequencing this library. I would just remake it.

06-21-2016, 12:50 AM	#11
windnature03 Junior Member Location: Hong Kong Join Date: Nov 2014 Posts: 3	how about sequencing the sample?? Were there any strange result or it is just a problem on bioanalyzer??