Equalizer kit versus qPCR for AmpliSeq libraries

[LindsayS]

I am curious if anyone is currently using the Equalizer kit from Life Tech to prepare their libraries for templation. If so, are you bulking multiple libraries together, and are you finding that it does a good job of having somewhat equal amounts of reads per library?

We frequently put 96 AmpliSeq libraries together on one 318 chip. Our method of quantitation is by qPCR. The Equalizer kit has been recommended to me on multiple occasions but I have not yet heard if it works well on multiplexed runs. We are at a point where qPCR is a huge bottleneck and we need to purchase another qPCR machine or figure something else out.

Any insight or user experience is welcomed.

[mikeg]

I would say to keep with your qPCR. I have tried the equalizer, and it is just too much of a black box for me. Doing 96 samples may be a bear, but you can rely on the qPCR data. Can't really say the same for the Equalizer!

[LindsayS]

Thanks Mike. I have come to the same conclusion. Our new solution is a 384-well qPCR machine. So excited!

[mikeg]

Hi Lindsay,

I see you are in Dallas? Where are you located?
I see you are putting 96 libraries onto one chip, but what are your libraries of?
Microbial?

[Xray1]

Hi there,

I would like to ask basically the same question as Lindsay.

For a larger NGS project with quite a lot of samples but relatively few amplicons (AmpliSeq), I find the idea of the Equalizer kit quite attractive. I would like to pool at least 32 samples per run, possibly even 64 or 96. Question is uniformity of course.

Does anyone here have some real life experience with it? Good or bad, any user experience is welcome.

[Élodie]

I will plan to use the equalizer too because I found some variations on the qPCR for Ampliseq DNA libraries (custom primers). So do you?
I tested several dilutions for one library and the calculated original concentration varies between dilutions. But I don't have this problem for other type of libraries (Exome, fragment plus)
Did you note the same thing?