ssDNA in sample from targeted exon capture affecting quantification

[vasvale]

Has anybody experienced this problem:?

I had too many clusters in a run and I’d like to ask if it ever happened to you: the DNA samples may have some single stranded DNA that is not quantified by the bioanalyzer nor Qubit (as they rely on intercalating dyes for dsDNA); I found out that using the kapa real time PCR kit (that picks up also ssDNA) the values are higher. The samples are 300 bp and in EB-tween 0.1%. After exon capture the elution from beads is alkaline and I wonder if that may be the reason why there some ssDNA.

[vasvale]

I usually load the flow cell for a GAIIx run at 9 pM.

[Heisman]

Are you using the Agilent kit? After elution the solution is alkaline, then you add in neutralization buffer, and then do PCR, which should cause everything to be double stranded.

[pmiguel]

Hi vasvale,
I think most people have this issue to one extent or another. Amplicons derived from adapter-dimers or primer-dimers can (and do) anneal to real (sample library) amplicons at the adapter sites they share.

Once annealed they will not be removed on the basis of molecular weight (gel cuts, Ampure isolation) unless these were to be done under strand denaturing conditions.

So even if you do a gel cut and determine that you have a clean band in the desired size range using an Agilent DNA chip, you can be fooled because the small amplicons are "hitchhiking" on the backs of your normal library molecules.

Then nearly all methods of determining the amplicon concentration of your sample will be confounded because they rely on the length determination done in the double stranded form.

I would recommend that you assay some of your libraries on an Agilient RNA chip after strand denaturing them:

http://seqanswers.com/forums/showthread.php?t=12523

If you see very large peaks below 150 nt then it is likely that some of them are hitchhiking small amplicons.

--
Phillip