qPCR's utility in nextgen library construction

[eca]

Does anyone use qPCR routinely in quantitating their nextgen libraries? If so, how difficult or easy it is and what's the cost per sample?

Thanks,
eca

[splicemaster]

qpcr nxtgen library contruction

hi eca,

did you ever find out what the costs were?

A

[jo_mason]

Hi Eca,
We routinely use qPCR to quantify our Illumina libraries, it takes alot of the guess work out of cluster generation. I'm not sure of the cost of the kits in the US, but out in Malaysia both the Kapa kit and the Agilent kit cost the equivalent of about $1-2 per reaction...of course as well as your actual sample there are standards to run, but it's not a huge cost compared to the cost of resequencing if you haven't got your cluster density right!
Best wishes,
Jo

[monad]

qPCR is essential if you want to maintain expected range of output. Make sure you include standard library that was prepared using same protocol for your library to be qPCR'ed and that was already sequenced so you know the target concentration for optimal clusters.

gDNA, mRNA, smallRNA, ChIP-seq, Exome enriched library....and insert size...they all perform differently for qPCR, so you will get better estimation of concentration only if you use library from the same protocol.

Regarding pricing, this is so negligible investment for the successful and maximum sequencing output.

[megalodon]

Standard for qPCR

Hi Monad,

for the longest time, I only quantitated my Illumina libraries with Picogreen and calculated the amount of library that was needed for cluster generation solely based on that. A few months ago, I started doing qPCR. It seems to be working very well for most of my libraries, however, it is less accurate when I do a qPCR using mRNA libraries. I know that the reason for this is most likely that we are always using Illumina's PhiX library as the standard, no matter what type of library we are trying to quantitate (mRNA, gDNA, ChIP...). In your post, you are saying that the standard needs to be a library that was prepared with the same protocol as the libraries to be qPCR'ed. However, this only works if you do know the accurate concentration of that library. How can you make sure that the concentration of your standard library is right?

[monad]

Hi megalodon,

As you know that the main reason we want to do qPCR was to measure the quantity of intact library, so we can maximize the output. I have seen enough that amplification efficiency of intact library from different type of library (gDNA and mRNA with different insert size, smRNA, ChIP) is not same. If you have library in your freezer that was made using sample protocol for the library you need to quantify, that would be ideal standard. As you pointed out, you need to know pM concentration used for flowcell and resulting sequencing yield. If you don't have such luxury, using standard (ex Kapa) is streamlined and easy way, and work close enough, but not necessary accurate if you need to dial in. Hope this helps.

[megalodon]

Hi Monad,

thanks a lot for your answer! I'm still not totally sure if I understand what I need to do to be able to use one of my own libraries as the standard for qPCR. Let's assume, I have never done a qPCR for mRNA libraries before but I do have a mRNA library which was already run on the HiSeq and could be used as the standard for other mRNA libraries that need to be qPCRed. Based on my quantitation results (Picogreen), I assume that the final concentration for cluster generation of that library was 9pM and the sequencing yield for that lane was 20Gb. I'm not totally sure how I would use that information to calculate how much of that library I would need to use for the qPCR standard dilutions? This is probably super easy and I'm just not getting it...

[Heisman]

Hi Monad,

thanks a lot for your answer! I'm still not totally sure if I understand what I need to do to be able to use one of my own libraries as the standard for qPCR. Let's assume, I have never done a qPCR for mRNA libraries before but I do have a mRNA library which was already run on the HiSeq and could be used as the standard for other mRNA libraries that need to be qPCRed. Based on my quantitation results (Picogreen), I assume that the final concentration for cluster generation of that library was 9pM and the sequencing yield for that lane was 20Gb. I'm not totally sure how I would use that information to calculate how much of that library I would need to use for the qPCR standard dilutions? This is probably super easy and I'm just not getting it...

You can use something else completely for a standard curve (to make sure the qPCR reactions themselves worked) and then treat your new library you are testing AND the one you've already sequenced as samples. Then you can just compare the two concentrations and figure out the idea loading concentration for your new sample.