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Old 09-28-2010, 02:00 PM   #1
ScottC
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Default 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.

Last edited by ScottC; 09-28-2010 at 03:00 PM.
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Old 09-28-2010, 03:41 PM   #2
Protaeus
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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.
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Old 09-29-2010, 11:57 AM   #3
antoniou
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I Agree. The Kappa kits have been great. our cluster numbers are very consistent since we started using the kit (6 months ago)
Eric
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Old 09-29-2010, 12:08 PM   #4
GW_OK
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We use and love the Kappa kit as well when used in conjunction with a Bioanalyzer for sizing.
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Old 09-30-2010, 06:06 AM   #5
NextGenSeq
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What else is there besides KAPA?
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Old 09-30-2010, 03:22 PM   #6
ScottC
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There's a few options... Kappa, many 'DIY' systems, Agilent, etc.
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Old 02-07-2013, 07:32 AM   #7
theward
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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!
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Old 02-07-2013, 08:02 AM   #8
kwaraska
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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.
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Old 02-07-2013, 04:51 PM   #9
cdalgard
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What are the primer sequences for this and is this for TruSeq libraries?
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Old 02-07-2013, 09:03 PM   #10
megalodon
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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.
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Old 02-13-2013, 07:24 AM   #11
theward
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Can I ask how you are sizing your libraries to calculate molarity? Is it the Bioanalyzer/Tapestation? If not, how do you size them?
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Old 02-14-2013, 05:10 AM   #12
pmiguel
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Quote:
Originally Posted by megalodon View Post
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.

--
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Old 09-30-2013, 12:02 AM   #13
oliwakun
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Arrow Qpcr

Quote:
Originally Posted by ScottC View Post
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
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Old 12-06-2013, 05:57 AM   #14
cement_head
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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
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Old 12-06-2013, 06:05 AM   #15
addyblanch
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The KAPA kit is a SYBR green mixture.
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Old 12-16-2013, 10:08 AM   #16
james hadfield
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We're just about to test BioRads ddPCR method. Will let you know how it goes!
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Old 12-17-2013, 08:55 AM   #17
austinso
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The ddPCR method is pretty good...but just thought I'd mention a clever twist:
Attached Files
File Type: pdf BTN_A_000114063_O_229541a.pdf (639.2 KB, 112 views)
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Old 01-16-2014, 11:56 AM   #18
JeremyDay
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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
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Old 01-17-2014, 07:02 AM   #19
pmiguel
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Quote:
Originally Posted by JeremyDay View Post
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.

--
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Old 11-06-2014, 10:13 AM   #20
DNA_Dan
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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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