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Old 06-07-2011, 07:47 PM   #7
sehrrot
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Location: USA

Join Date: Jul 2010
Posts: 58
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Dear ZAAB

I've also seen this issue for long time. Please read my recent post, which I saw same thing on bioanalyzer. http://seqanswers.com/forums/showthread.php?t=11886

I also assume, as others, that it might be a problem of long ligation incubation. When I firstly made Truseq library, I did 30 minute incubation as I thought it's too short as well as old PE library protocol recommends. But I saw very definite dimer on bioA profile and gel. (I also used to assume that it might be a secondary problem in the bioA but bioA reagent includes DMSO to prevent 2nd structure and gel showed big fragment, which means that it's not simply random hybridized fragment.)


As I wrote in my recent post in this forum, I did PCR optimisation and got conclusion, which I should use around 3 ng PCR input to avoid this big fragment issue. I guess if we just use whole genomic sequencing, it's not gonna be a problem as bridging amplficiation will sort out big fragments by size. But if we use exome sequencing, which needs hybridisation with probes, it might be a problem that probes can bind to big fragment more than aimed fragment (actually, I've seen this one and probes was bound to big fragment so overall capture efficiency reduced).

I still don't know what exactly this big fragment is. I cut this big fragment and did qPCR. the result was really lower molarity than I put. Also, on the sequencer, it didn't make enough cluster generation. So, I guess truseq library prep provides very strong ligation step, as a result, some fragment can be ligated randomly to other library if ligation time is longer.
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