Same adapter sequence on both ends of fragments
Hi all,
I am preparing a DNA library for Miseq and ran into problems with the library adapters (Illumina and Nextflex). After ligating the adapters to my A-tailed fragments and doing the PCR, I cloned some the fragments and realized that most of them have the same adapter sequence on both ends. To explain myself better, here is what I mean by that:
correct: >Adapter1>----XXXXX---->Adapter2>
in my case: >Adapter1>----XXXXX----<Adapter1<
> = orientation
In theory this should be impossible due to the T-overhang in the ds-adapter and the A-tailing of the fragments. However, this is what I get. I am not sure if the issue is the ligation or the PCR. I already tried to repeat the A-tailing, to use different adapters (Illumina & Nextflex) and perform the PCR with less DNA (1:15). Right now I am going even further down with the amount (1:150) to see if that helps. Otherwise I thought about cloning and sequencing the ligation products before the PCR (after a quick denaturation).
Did anyone of you ever have similar issue and could give me some advice? I would very much appreciate it!
Thanks a lot.
Philipp
I am preparing a DNA library for Miseq and ran into problems with the library adapters (Illumina and Nextflex). After ligating the adapters to my A-tailed fragments and doing the PCR, I cloned some the fragments and realized that most of them have the same adapter sequence on both ends. To explain myself better, here is what I mean by that:
correct: >Adapter1>----XXXXX---->Adapter2>
in my case: >Adapter1>----XXXXX----<Adapter1<
> = orientation
In theory this should be impossible due to the T-overhang in the ds-adapter and the A-tailing of the fragments. However, this is what I get. I am not sure if the issue is the ligation or the PCR. I already tried to repeat the A-tailing, to use different adapters (Illumina & Nextflex) and perform the PCR with less DNA (1:15). Right now I am going even further down with the amount (1:150) to see if that helps. Otherwise I thought about cloning and sequencing the ligation products before the PCR (after a quick denaturation).
Did anyone of you ever have similar issue and could give me some advice? I would very much appreciate it!
Thanks a lot.
Philipp