Problem with TruSeq Custom Amplicon Libraries

HelenaSC · 05-10-2016, 01:45 AM

Hi all!
We need some help with TruSeq Custom Amplicon Low Input libraries 
We have been processing samples (fresh and FFPE) for a while using this Kit and everything worked fine. Before starting the protocol, we always perform the qPCR to check the quality of the samples and we choose the DNA input as described in the userguide.

Lately, we are experiencing some problems that we don't understand and that seem to be "hazardous", we are not able to see what's going on…the issue is that we are getting very strange libraries's profiles on the Bioanalyzer 2100 (attached images). Some libraries are not working, others are working ok, and some of them are having some strange peaks before the library (they seem like a kind of "horns") that we can't get ride off, despite purifying the samples again and again….Moreover, it seems that these libraries are carrying something that make the bioanalyzer run not properly, dirty or similar…

Any one has experienced this issue? Any idea of what could it be? The kit is the same that used to work fine some days before, and the controls are working ok randomly!

Thanks a lot in advance!!
Presentación1.jpg

Markiyan · 05-10-2016, 03:05 AM

Those traces resemble the "broad peaks" artefacts from Sanger sequencing machine, which were a result of sequencing reaction working too well (or too long injection time), which basically causes a congestion in the capillary, causing loss of size separation.

If you check those traces on the time scale, you should see, that they take a bit longer to come out, than normally.

Also you can see from the makers, that the sample concentration is ~10X higher, than in the first case. So try rerunning your libraries with 10x or more dilution, and see what you get.

Also some sort of proteins/etc bound to DNA may cause similar "Gel - shift" pattern.

LAO · 07-02-2017, 10:52 AM

Quote:

Originally Posted by HelenaSC

Hi all!
We need some help with TruSeq Custom Amplicon Low Input libraries 
We have been processing samples (fresh and FFPE) for a while using this Kit and everything worked fine. Before starting the protocol, we always perform the qPCR to check the quality of the samples and we choose the DNA input as described in the userguide.

Lately, we are experiencing some problems that we don't understand and that seem to be "hazardous", we are not able to see what's going on…the issue is that we are getting very strange libraries's profiles on the Bioanalyzer 2100 (attached images). Some libraries are not working, others are working ok, and some of them are having some strange peaks before the library (they seem like a kind of "horns") that we can't get ride off, despite purifying the samples again and again….Moreover, it seems that these libraries are carrying something that make the bioanalyzer run not properly, dirty or similar…

Any one has experienced this issue? Any idea of what could it be? The kit is the same that used to work fine some days before, and the controls are working ok randomly!

Thanks a lot in advance!!
Attachment 4328

Hello Helensc
Did you resolve the issue with your TruSeq custom amplicon libraries? I am seeing the same problem. Any information would be much appreciated. Thanks!

05-10-2016, 01:45 AM	#1
HelenaSC Member Location: Spain Join Date: Jun 2013 Posts: 21	Problem with TruSeq Custom Amplicon Libraries Hi all! We need some help with TruSeq Custom Amplicon Low Input libraries  We have been processing samples (fresh and FFPE) for a while using this Kit and everything worked fine. Before starting the protocol, we always perform the qPCR to check the quality of the samples and we choose the DNA input as described in the userguide. Lately, we are experiencing some problems that we don't understand and that seem to be "hazardous", we are not able to see what's going on…the issue is that we are getting very strange libraries's profiles on the Bioanalyzer 2100 (attached images). Some libraries are not working, others are working ok, and some of them are having some strange peaks before the library (they seem like a kind of "horns") that we can't get ride off, despite purifying the samples again and again….Moreover, it seems that these libraries are carrying something that make the bioanalyzer run not properly, dirty or similar… Any one has experienced this issue? Any idea of what could it be? The kit is the same that used to work fine some days before, and the controls are working ok randomly! Thanks a lot in advance!! Presentación1.jpg Last edited by HelenaSC; 05-10-2016 at 01:47 AM. Reason: wrong title

05-10-2016, 03:05 AM	#2
Markiyan Senior Member Location: Cambridge Join Date: Sep 2010 Posts: 116	Try lowering the concentration by 10x when loading the bioanalyzer. Those traces resemble the "broad peaks" artefacts from Sanger sequencing machine, which were a result of sequencing reaction working too well (or too long injection time), which basically causes a congestion in the capillary, causing loss of size separation. If you check those traces on the time scale, you should see, that they take a bit longer to come out, than normally. Also you can see from the makers, that the sample concentration is ~10X higher, than in the first case. So try rerunning your libraries with 10x or more dilution, and see what you get. Also some sort of proteins/etc bound to DNA may cause similar "Gel - shift" pattern.