Unsual peak in a RAD seq library

[laura.benestan]

Hi all,

I'm preparing RAD sequencing library with a protocol inspired by Etter et al, except that I use Amplicon Beads instead of a gel.
Before sending to the sequencing platform I make a High sensitivity DNA chip (AGilent machine) to check the size of my fragment (normally between 300-800 base pairs).
On most of my samples I got a big peak at about 3000 base pairs, something that I don't understand.

Firts I thought it was the beads. But, this peak appear only after the PCR step, it is not there before PCR or either after sonication.
Is there anyone here that had the same problem ?
Also, some others people had the same problem than I with a different protocol, the GBS one (Elshire et al), but the same machine (Agilent).

Thanks for your help,

Laura

[JackieBadger]

I havent read that specific protocol but could hazard a guess perhaps. Do the large fragments occur after digestion?
If so it could be large chimera formation via your sticky ends (if your protocol produces them). I know that this is an issue in the genotyping by sequencing protocol, and in ddRAD. Just modify the protocol and size select as in the ddRAD protocol. Then when you align to a genome you should know the distance between your paired ends. If this is for eg much bigger than expected, this is probably due to chimera formation.

I am guess taking a guess here

[pmiguel]

Hi all,

I'm preparing RAD sequencing library with a protocol inspired by Etter et al, except that I use Amplicon Beads instead of a gel.
Before sending to the sequencing platform I make a High sensitivity DNA chip (AGilent machine) to check the size of my fragment (normally between 300-800 base pairs).
On most of my samples I got a big peak at about 3000 base pairs, something that I don't understand.

Would you post the image, please?

--
Phillip

[laura.benestan]

Thanks for your quick answer.
Enclosed, there is the Bioanalyser profil displaying an unusual peak at about 3000 bp.


Those fragments doesn't appear after digestion. We made an agarose gel yesterday and there were no detectable amount of such fragment at 3000bp. It seems to be an artefact....but we still don't understand why this fragment appears only after PCR.

At the beginning, we also thought that was due to the sticky ends. However, the size of this big fragments is always the same (about 3000bp) whereas we should expect some size variation in this case.