CPB correction with emPCR format change

[454User]

Hi there,

It is my understanding that you generally need to adjust the CPB value of the emPCR when going from titration SV format to the production LV format to correct for the greater enrichment % of the latter. Do you all have a rule of thumb value that you use for this decrease (E.g. 0.25 CPB less for LV)?

Thank you!

[ChickSeq]

Hello,

We make up our emulsions for Titanium Lib-L LV kits at the same cpb that yields between 8-10% recovery for the SV titration. This seems to consistently give us good results without having to adjust the end cpb. With the old FLX kits, we used to have to adjust the cpb down, but since the switch to Ti Lib-L, this hasn't really been a problem.

However, we've noticed that with the Lib-A kits, the post-enrichment percent recovery for LV has been all over the place in comparison with the SV titration recoveries. Has anybody else noticed this happening with the Titanium Lib-A kits in particular?

Thanks,

Jen

[454User]

Thank you very much for your input, Jen. It must be a lab-specific problem that I tend to see higher enrichments even with the Ti chemistry.

I'm afraid that I can't be much of a help wrt the LV Lib-A kits as all of my amplicon customers have only wanted unidirectional sequencing thus far.

-J

[Zaag]

We have the same with LV, so we tend to use MV or SV.

Don't know about Lib-A kits though, sorry.

[454User]

We have the same with LV, so we tend to use MV or SV.

Don't know about Lib-A kits though, sorry.

Thanks for your reply, Zaag. I have also switched over to exclusively SV... it is more laborious but I am willing to sacrifice the time and effort for the gain of reproducibility.

I also do not have much experience with the Lib-A kits as my customer have only requested uni-directional sequencing up to this point.

-J