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-   -   Primerdimer/Short Product Bias on the MiSeq (http://seqanswers.com/forums/showthread.php?t=41095)

Vinz 02-21-2014 01:28 AM

Primerdimer/Short Product Bias on the MiSeq
 
Primerdimers are more efficient in cluster formation than longer products.
Our data suggests that the extent of this preference can be influenced by sample preparation after (!) cleanup. Anyone has a guess or even knowledge what step / how one could influence the ratio of primerdimer clusters versus normal size products?
Denaturing conditions?
Handling times?
Temperatures?
HT1 buffer?

Background: We experienced that the rate of primerdimer rate is changing over time. In particular we have one example of 8 pools of samples that were cleaned up together but put on different runs. They show a marked difference in primerdimer rate that can not be explained by the nature of the samples.

nefertitichen 02-21-2014 07:49 AM

I would try to get rid of the primer dimers with AMPure beads before pooling for sequencing.

thomasblomquist 02-21-2014 09:45 AM

Post-PCR cleanup of multiplex amplicon library prep is almost a given. Ampure beads, gel electrophoresis and band cut out, column exclusions, etc, are all decent options for Illumina library prep clean up and removal of primer dimers.

This is not the same thing as universal multiplex pcr library amplification which has a single primer that amplifies all products that have a common adapter attached on each end.

That being said, annealing temperature and time is key to minimization primer dimer formation, as well as any sort of off-target priming. There is a direct correlation between the specificity of the priming and annealing temperature. Conversely there is an inverse relationship with cycle-to-cycle yield/efficiency. Another consideration is to back off of primer concentration if specific primers are giving non-specific dimerization products.

Remember, PCR is a COMPLEX enzymatic reaction, with a lot of potential side reactions if you don't optimize the kinetics favorably for the desired product formation.

Regards,

-Tom Blomquist

snetmcom 02-21-2014 04:03 PM

Pippin Prep
Ampure Cleanup
EGel

It usually takes 2 rounds of cleanup to fully remove most byproducts.
All of these options will be easier than trying to configure cluster generation.


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