Tweet
ChIP-Seq library prep; trailing, primer dimer help?
Hi,
I've prepared ChIP-Seq libraries using NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) along with the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645L). The samples will be sequenced on Novaseq 6000 S4, PE150, 30 million reads / sample.
When I ran them on a bioanalyzer, they had trailing, primer dimer and adaptor dimer contamination. I did 1 PCR cycle followed by beads clean up and this helped in removing the adaptor dimers.
I'm attaching a photo of the current bioanalyzer trace.
Would it be ok to proceed with sequencing these libraries as they are?
As far as I understand, the primer dimers will not be sequenced, since they don't contain any adapters. I'm not quite sure about the effect of the trailing that we see.
If that's not ok, what are the consequences and what can I do?
I was thinking of doing a size selection with the beads, but I'm not sure if that would result in a great loss of the libraries. Also, from what I read, the trailing could be carry over beads, if that's true and the libraries are placed on a magnet for a couple of minutes and the solution aspirated and collected, would that help?
Thank you in advance.
I've prepared ChIP-Seq libraries using NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) along with the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645L). The samples will be sequenced on Novaseq 6000 S4, PE150, 30 million reads / sample.
When I ran them on a bioanalyzer, they had trailing, primer dimer and adaptor dimer contamination. I did 1 PCR cycle followed by beads clean up and this helped in removing the adaptor dimers.
I'm attaching a photo of the current bioanalyzer trace.
Would it be ok to proceed with sequencing these libraries as they are?
As far as I understand, the primer dimers will not be sequenced, since they don't contain any adapters. I'm not quite sure about the effect of the trailing that we see.
If that's not ok, what are the consequences and what can I do?
I was thinking of doing a size selection with the beads, but I'm not sure if that would result in a great loss of the libraries. Also, from what I read, the trailing could be carry over beads, if that's true and the libraries are placed on a magnet for a couple of minutes and the solution aspirated and collected, would that help?
Thank you in advance.