What's your favourite qPCR kit for Illumina library QC?

[ScottC]

Hi All,

Just wondering which kits (if any) people are using for QC-ing Illumina libraries? From what I've read so far, people seem to like the Kappa kits.

Cheers,

Scott.

[Protaeus]

I concur with the rumors you have heard - the Kapa kit has given me consistent and reproducible concentrations. I recommend running a qPCR on your pools as an extra QC check if you are multiplexing.

[antoniou]

I Agree. The Kappa kits have been great. our cluster numbers are very consistent since we started using the kit (6 months ago)
Eric

[GW_OK]

We use and love the Kappa kit as well when used in conjunction with a Bioanalyzer for sizing.

[NextGenSeq]

What else is there besides KAPA?

[ScottC]

There's a few options... Kappa, many 'DIY' systems, Agilent, etc.

[theward]

From reading this and other threads most people prefer the KAPA kits to run qPCR for library quantification. Can anyone tell me why these kits are superior to the SYBR green kits as my understanding is they're more expensive? If it to do with the standards being normalised could you not just use your own library to generate a standard curve with the SYBR green kits?
Any advice most welcome!

[kwaraska]

We use the Kappa enzyme mix with our own primers and use PhiX as a standard curve.

The main reason we don't use the Quant Kit itself is it is far more expensive than buying the enzyme and primers seperately. That being said, we are a core so we are generally QCing 20 or more samples at a time so the standard curve is but a small component of the cost.

[cdalgard]

What are the primer sequences for this and is this for TruSeq libraries?

[megalodon]

Check Illumina's qPCR protocol. The primer sequences are listed in there.

We are also using the Kapa master mix together with our own primers and PhiX as standard for qPCR. While we are also getting fairly consistent results, our qPCRs tend to overestimate the concentration of our libraries, especially for RNA-seq libraries. Has anyone else seen this? We also tried the Kapa primers and standards but got similar results.

[theward]

Can I ask how you are sizing your libraries to calculate molarity? Is it the Bioanalyzer/Tapestation? If not, how do you size them?

[pmiguel]

Check Illumina's qPCR protocol. The primer sequences are listed in there.

We are also using the Kapa master mix together with our own primers and PhiX as standard for qPCR. While we are also getting fairly consistent results, our qPCRs tend to overestimate the concentration of our libraries, especially for RNA-seq libraries. Has anyone else seen this? We also tried the Kapa primers and standards but got similar results.

Just a word of warning: we have found the Illumina phiX libraries to be variable in concentration. Differences observable even using a high sensitivity chip.

We get lower batch to batch variability using Kapa's own standard.

--
Phillip

[oliwakun]

Qpcr

There's a few options... Kappa, many 'DIY' systems, Agilent, etc.

Yeah, you should try Agilent. Got the best kits at affordable prices. Ask to speak to Abraham (Bioreagents Specialist) and he can advise you on what to get. Company number is 0845 712 5292 Option 3 then 1

[cement_head]

Hello,

We've had excellent results with the KAPA kit (universal) recommended by Illumina. It's a tad expensive ($575) - but it enables you to really get very accurate potential clustering QC data.

- Andor

[addyblanch]

The KAPA kit is a SYBR green mixture.