problem with the ligation.

[induivain]

Hi all.
I had a problem, I think with the ligation step problably.
The fragmentation with covaris went well, i had a mean of 225bp fragments.
After ligation, the fragments have increased in size.
After amplification , i check 4, 5 and 6 cycles, the fragments have increased in size , but the shape of the curve is extended to upper Marked (1500bp)
I contacted with the techicall support and they suggested an extra cleaning with beads, but the result remains the same.
I use a sure select XT

Does someone have this problem?.

In the attachment you can see the results of the Bioanalyzer.

All help is welcome

[pmiguel]

What method/kit are you using?

In any case, it look like you are probably just seeing a phenomenon caused by the ectopic annealing of adapter creating "bird nesting", "daisy chaining" and/or "bubble products".

You can generally avoid them by doing fewer cycles of enrichment PCR. (But, if you are using TruSeq adapters from a TruSeq kit, you must do at least one cycle to "break the fork", evidently). But they do not tend to cause a problem for sequencing because all the ectopic annealing is removed when the samples are denatured prior to cluster PCR. So your libraries are probably fine.

--
Phillip

[induivain]

Many thanks for your reply Phillip
I am using SureSelect XT Target enrichment System for illumina Paired-end sequencing Library.

In my case i have the problem in the ligation of the sample preparation prior to hybridization.

With this "bird nesting" phenomenon my doubt is if i can go on with the hybridization because maybe the capture is not efficient.

I repeated 4 samples more , and i had the same traces in the high size of DNA, always after the ligation and in previous steps without bird nesting phenomenon.