strange library results

beetle_doc · 03-07-2012, 11:41 AM

I have run 3 sample using TruSeq genomic prep using both gel and non gel based methods. The resulting libraries when run on a bioanalyzer all looked like lane 6 of the attached document. Other libraries from the same order looked fine. The input DNA was of sufficient quantity and looked fine on a gel. I have also seen this pattern with one TruSeq RNA library prep sample. Has anyone else seen this?
Thanks,
Mike

pmiguel · 03-08-2012, 04:21 AM

My first guess would be that they are the TruSeq Control fragments. These are an optional component of both RNA and DNA prep kits. They have a variety of sizes and sequences. The HiSeq Control Software (HCS)/Real Time Analysis (RTA) software recognizes their sequence and their relative proportions are plotted in a pane of the Sequence Analysis Viewer (SAV).

I don't use them, so I can't tell you for sure. Under normal circumstances I think they are likely at too low of a concentration to be detectable amongst the background of your sample. But in cases where scant sample is present, they might become alarmingly visible. Could be my imagination, but I think I can see faint peaks of similar sizes in lane 5, for example.

The BioAnalyzer individually scales each lane by default. If you are viewing your data via the Agilient 2100 Expert Software you can turn this behavior off by clicking on the "Gel" tab and the sub-category "Scaling Mode" to choose "Global Scale". I am guessing that if you do this, then the peaks you see clearly in lane 6 will fade into the background. Which indicates the issue is that there is no (or almost no) sample there.

--
Phillip

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03-08-2012, 04:21 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	My first guess would be that they are the TruSeq Control fragments. These are an optional component of both RNA and DNA prep kits. They have a variety of sizes and sequences. The HiSeq Control Software (HCS)/Real Time Analysis (RTA) software recognizes their sequence and their relative proportions are plotted in a pane of the Sequence Analysis Viewer (SAV). I don't use them, so I can't tell you for sure. Under normal circumstances I think they are likely at too low of a concentration to be detectable amongst the background of your sample. But in cases where scant sample is present, they might become alarmingly visible. Could be my imagination, but I think I can see faint peaks of similar sizes in lane 5, for example. The BioAnalyzer individually scales each lane by default. If you are viewing your data via the Agilient 2100 Expert Software you can turn this behavior off by clicking on the "Gel" tab and the sub-category "Scaling Mode" to choose "Global Scale". I am guessing that if you do this, then the peaks you see clearly in lane 6 will fade into the background. Which indicates the issue is that there is no (or almost no) sample there. -- Phillip