adapter dimer

[yfondyfe]

Hi everyone,

I am having issues with adapter dimer in my RNA preps for sequencing. I have read about the dimer eliminator but it is expensive. Is there another method to deal with this or is there a kit that does better? I have read about the netflex small RNA kit (BIOO scientific), however, is there something I can do without buying some other kit or LNA?

[nucacidhunter]

If you provide specific information such as the kit used for library prep, the issues that you see and measures taken o alleviate it that has not worked well, you are more likely to get useful responses. There are many methods and kits for RNA library prep and general advice might not be helpful for your adapter-dimer issues.

[yfondyfe]

We are using the small RNA library kit from NEB. We are doing PAR-CLIP ad then adapting the RNA for illumina sequencing. After PCR amplification, we see a lot of adapter dimer in our PCR samples. In fact, it pver-rides the samples. I have read about dimer eliminator but was wondering if there is a simpler way to avoid this adapter dimer product or even reduce it. I think also that increasing the starting RNA sample amount might be easier but if anyone has done PAR-CLIP before, the resulting RNA is very low.

[nucacidhunter]

Dimer eliminator will not help to reduce adapter-dimmer formation when using NEB small RNA kit. Annealing of RT primer (after 3’ adapter ligation) to un-ligated 3’ adapter would effectively reduce primer-dimer formation by transforming 3’ adapter in to a double-stranded DNA that is not a substrate for T4 RNA ligase used for 5’ adapter ligation. I wonder what your RNA size range is and if you have any RNA and prepped library Bioanalyser trace. Primer-dimer that you observe might have different origin (it is less likely product of ligations between 3’ and 5’ adapters) and that can be identified by examining primer-dimer sequences. Cutting library fragments with expected size from gel is one way to reduce primer-dimer reads in sequencing data. It might also be possible to reduce them by modifying library prep protocol.