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-   -   What's your favourite qPCR kit for Illumina library QC? (http://seqanswers.com/forums/showthread.php?t=7064)

ScottC 09-28-2010 02:00 PM

What's your favourite qPCR kit for Illumina library QC?
 
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 09-28-2010 03:41 PM

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 09-29-2010 11:57 AM

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 09-29-2010 12:08 PM

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

NextGenSeq 09-30-2010 06:06 AM

What else is there besides KAPA?

ScottC 09-30-2010 03:22 PM

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

theward 02-07-2013 07:32 AM

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 02-07-2013 08:02 AM

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 02-07-2013 04:51 PM

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

megalodon 02-07-2013 09:03 PM

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 02-13-2013 07:24 AM

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 02-14-2013 05:10 AM

Quote:

Originally Posted by megalodon (Post 95884)
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 09-30-2013 12:02 AM

Qpcr
 
Quote:

Originally Posted by ScottC (Post 26225)
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 12-06-2013 05:57 AM

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 12-06-2013 06:05 AM

The KAPA kit is a SYBR green mixture.

james hadfield 12-16-2013 10:08 AM

We're just about to test BioRads ddPCR method. Will let you know how it goes!

austinso 12-17-2013 08:55 AM

1 Attachment(s)
The ddPCR method is pretty good...but just thought I'd mention a clever twist:

JeremyDay 01-16-2014 11:56 AM

I know this thread is a bit older now, but I am interested in other alternatives as well. Kapa is darn expensive, but IS consistent. I think I'll need to play with different concentrations of primer, along with different kits, including the kapa universal Fast Sybr to find an alternative Sybr green kit to reduce cost and keep consistency.

One thing I am wondering: Does anyone have a clue as to how the Kapa standards are not degrading at such low concentrations (lowest is 0.0002pM)? If i remember correctly, DNA under 1nM is susceptible to degradation even in -20 storage. My concern is that, even if I amplify my own library, kapa standard, or PhiX standard, that it will degrade if I keep stocks of the lower concentrations. I can do a serial dilution, but I'd rather be able to keep premade stocks in a PCR strip so that I can use a mutlichannel for qPCR setup. Do you think there are any modifications? I tried to amplify a Lifetech taqman standard one time and was unable to get a clean product, and I wonder if it was due to the design of it, rendering it incapable of being amplified.

Would it make sense to add a phosphorothioate bond into my primers if I attempt to amplify either the kapa or PhiX in order to reduce degradation? Do those bonds protect only oligos, and not an entire amplicon?

Has anyone else come up with a reliable method for quantification of a pooled library other than using kapa kits?

Thanks for your opinions!!
Jeremy

pmiguel 01-17-2014 07:02 AM

Quote:

Originally Posted by JeremyDay (Post 129846)
One thing I am wondering: Does anyone have a clue as to how the Kapa standards are not degrading at such low concentrations (lowest is 0.0002pM)? If i remember correctly, DNA under 1nM is susceptible to degradation even in -20 storage.

"susceptible to degradation"? What on earth do you mean?

Not sure who told you this, but it sounds like some sort of folklore invented to explain why yields, etc. are so poor (percentage-wise) when the amount of sample is very low.

I have a better folktale for you: the reason this happens is that everything has a binding capacity for macromolecules. For plastics this binding capacity is probably mostly fairly low -- such that at ug levels, it is not discernible. But the smaller amount of a macromolecule you process, the more noticeable loss to this sort of binding will be.

Mitigating against these sorts of losses would involve using low bind plastic and/or spiking your samples with a detergent.

--
Phillip

DNA_Dan 11-06-2014 10:13 AM

Anyone have any feedback on the Biorad QX200? Does it work as advertised for NGS quantitation or is this just "smoke and mirrors"? Seems like the next logical step to take.


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