Question: Help with NEBNext RNA Library Prep: Samples with different amounts of PCR amplification!
gravatar for jlcoffin3
6 weeks ago by
jlcoffin30 wrote:

Hey folks,

I'm using a NEBNext Ultra-directional RNA Library Prep kit for an RNA-seq experiment. I've been using 12 cycles of PCR during the amplification step and it has been working great, but I realized I accidentally used 15 cycles instead of 12 on the last run. Does anyone know of a way to control for differences in PCR amplification once I get the reads back? I unfortunately can't afford to redo the library prep for these samples. Any help would be amazing!



rna-seq • 150 views
ADD COMMENTlink written 6 weeks ago by jlcoffin30

Hello jlcoffin3!

It appears that your post has been cross-posted to another site:

This is typically not recommended as it runs the risk of annoying people in both communities. You should at least remark where you've already posted this question.

Also I'm not sure whether this topic is bioinformatic related. So seqanswers might be the better place to put your question.

fin swimmer

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by finswimmer4.4k

No, there is no way to deal with this. In theory, the sample should simply be more concentrated but PCR bias will do its part in unequally amplifying some reads more than others. I would see if after sequencing the sample reasonable clusters in a PCA with the other replicates and shows good correlation. If so, proceed as planned. 3 cycles are not a catastrophy. There are RNAseq-like experiments where you do far more PCR and they still produce results that are good ;-)

ADD REPLYlink written 6 weeks ago by ATpoint6.5k
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