127bp adaptor dimer

[chipgal]

Hi all,

I ran Bioanalyser on the PCR products of my RNA-Seq library prep, and there are adaptor dimers contamination in all my samples. Some are more serious (sample 1) than the others (sample 3) as the starting amount of RNA varies among my samples. Pls see attached pic.

Should I do a AMPure beads purification on all? Or should I do a gel purification instead? Do all my samples (sample 1,2 and 3) all warrant 2nd purification in order to get rid of the adaptor dimers? I am going to run a multi-plex Hi-Seq run, 8-12 samples per lane, so it will be ~25M per sample.

Thanks a lot!

[bunce]

Hi Chipgal,
We use long fusion primers in our workflows and they can be tough to get rid of. As a first option I would try ampure clean-ups - I suspect an ampure ratio of 1.2 might do the trick.... it does depend on input DNA and a few other factors so you could titrate. There are some threads on this if you search... for example check out http://www.keatslab.org/blog/pcrpuri...mpureandsimple

Cheers, Mike

[weigrc]

Hi Chipgal,

We see the adapter dimers sometimes. Usually do 1X AMPure beads purification to get rid of the dimers, as it affects the number of sequencing reads a lot.

Thanks,
Wei