[TonyBrooks]

I just saw exactly the same thing. I've run hundreds of samples on our GAIIx's but we thought we'd switch to qPCR after upgrading to the HiSeq.
I used Qubit values to normalise to what I thought was 10nM. When I ran those on qPCR I was getting anything from 5nM to 30nM.
I used both the standard in the KAPA kit and a homebrew (from Illumina PhIX). Both standard curves had R2>0.999. The efficiency of the PhiX curve was 104%, but the KAPA had much more variable efficiency (80-120%).
The kit standard gave me ~30% lower concentration values than the PhiX.
I went with the PhiX as I'd run that on the HiSeq during training so I knew the expected cluster density was good at 17pM, I didn't want to over cluster and there were strange efficiency values for the Kapa curve.
However, when I sequenced, the density was much lower than expected (~500k/mm2). The KAPA standards would have given more accurate quantification.
Finally, to compound matters, we had one sample in a multiplex pool give twice the number of reads than we would expect if everything was equimolar. So it looks like it was under-quantified by qPCR.
So although qPCR seems to be better than Qubit, it's not without its problems.
I would be interested if anyone has some ideas on how to optimise to increase accuracy too.

[Genquest]

Hi HGENETIC,

Thank you for your reply!!I feel relieved to know that others also have experienced such problem).
What happened in my case was just the reverse....We took the qPCR concentration for cluster generation (for V3 flow cell on hiseq2000) assuming that the qPCR would give accurate results and landed with under clustering.

I guess we just cannot take the qPCR results straightforward...have to see the trend.Need some advise on optimization!!
Can anyone help please??

Thanks in advance!!

[HGENETIC]

Hello everyone,
I am pretty new to this forum and stumbled on this post as I am facing problem with qPCR quantification method
1.Has anyone encountered with, a higher qPCR concentration than the estimated concentration by pico green??
2. Also if assumed that qPCR is more accurate(w.r.t pico green) as it would give conc. of ssDNA ligated with adapters,then the cluster generation should be accurate with qPCR conc. but it doesn't happen so...the flow cell (V3,Hiseq 2000) remains under clustered.Can any one think of possible reasons??
3. This trend seems to change with every library.Is absolute quantification the right approach for Sybr green qPCR method?Also I am using the PhiX control provided by Illumina as my standard.
4.Do people take the "exact concentration of libraries" as determined by qPCR for cluster generation or they compare it with pico green to see the relative trend of concentration?

Would appreciate if any of the queries are resolved!!
Thanks in advance

Hi Genquest,

we had a similar situation to yours in that our qPCR results gave higher DNA concentration than our picogreen (see post above for qPCR kit used). However, when ouor sequence provider ran a qbit the results were more in line with our picogreen and so we decided to use that concentration for cluster generation. When the samples were run we had a very high cluster density and it looked like the qPCR results may of been the most accurate, luckily we still got good data but I can't explain why the two methods gave the results they did and we haven't run anymore samples so I can't comment on whether this was a chance result. My advice would be use one method (preferably qPCR), optimise it and stick with it. Cheers H

[Genquest]

Library qualification by qPCR

Hello everyone,
I am pretty new to this forum and stumbled on this post as I am facing problem with qPCR quantification method
1.Has anyone encountered with, a higher qPCR concentration than the estimated concentration by pico green??
2. Also if assumed that qPCR is more accurate(w.r.t pico green) as it would give conc. of ssDNA ligated with adapters,then the cluster generation should be accurate with qPCR conc. but it doesn't happen so...the flow cell (V3,Hiseq 2000) remains under clustered.Can any one think of possible reasons??
3. This trend seems to change with every library.Is absolute quantification the right approach for Sybr green qPCR method?Also I am using the PhiX control provided by Illumina as my standard.
4.Do people take the "exact concentration of libraries" as determined by qPCR for cluster generation or they compare it with pico green to see the relative trend of concentration?

Would appreciate if any of the queries are resolved!!
Thanks in advance

[sehrrot]

I agree with Lee

we used KAPA, this is the best option for cluster density. We've succefully used it for V2 and V3 SBS kit.

[HGENETIC]

We've always used picogreen and found it matches well with the qbit results that our sequence service provider uses to make library dilutions before running. However, after some curious results we are going to try the Agillent NGS QPCR system as this is supposed to be more accurate, just have to wait to see how the results tie up.

[niceday]

to ktabbada

If you are seeing qPCR results that are 10% of your bioanalyser or Qubit readings and you successfully clustered using the qPCR concs then you should have your bioinformaticians scan the data to see if you have low complexity library.

Also are you making PCR or nonPCR libraries?

With PCR after adapter ligation properly ligated material should drown out incorrectly ligated library and your bioanalyser/Qubit should be close to your qPCR conc. If you see a big difference between bioanalyser/Qubit and qPCR you might have low ligation efficiency or poor PCR amplification.

On another note I have seen qPCR data higher than both ubit and bioanalyser. I always use qPCR and cluster accordingly.

[pmiguel]

do you expect to detect ssDNA in DNA high sensitivity assay at all? isn't it a dsDNA binding dye used there?

Regardless of what my expectations were, we have run the ssRNA ladder on a DNA high sensitivity chip. We did detect it.

See:

http://seqanswers.com/forums/showthread.php?t=12852

for details.

I should add that when we asked Agilent (prior to running this chip) what results we should expect when running single stranded molecules on a double stranded chip, we were told that the chip was not appropriate for that purpose. (Thanks for nothing, Agilent...)

--
Phillip

[kulukulas]

(1) ssDNA runs slower than one might expect on a High Sensitivity DNA Bioanalyzer chip. (Probably also on other DNA Bioanalyzer chips). Run a lane of nano RNA ladder on your DNA chip to get an idea. Anyway, this can be confusing under some circumstances.

do you expect to detect ssDNA in DNA high sensitivity assay at all? isn't it a dsDNA binding dye used there?

[pmiguel]

The Qubit is specific for dsDNA. If you sample contains ssDNA with adapter ends, it will be detectable by qPCR but not Qubit. (Note: it will also form clusters, which makes qPCR more accurate for quantification.)

In addition to ssDNA amplicons in your library, adapter dimers (ssDNA or dsDNA) could also be a source of higher molar concentration readings from qPCR versus fluorimetry.

Adapter dimers (zero or very short insert amplicons) will form clusters but will be detected at different sensitivities by different QC assay methods. Probe-based qPCR would likely detect them accurately. SYBR-green based qPCR might underestimate their molar concentration.

As detailed up-thread, Agilent 2100 Bioanalyzer results will detect non-amplicons (one or zero adapter ends) the same as amplicons -- other than the (probably minor) shift in length. Other confounding aspects include:

(1) ssDNA runs slower than one might expect on a High Sensitivity DNA Bioanalyzer chip. (Probably also on other DNA Bioanalyzer chips). Run a lane of nano RNA ladder on your DNA chip to get an idea. Anyway, this can be confusing under some circumstances.

(2) dsDNA runs faster than one might expect on RNA (single-stranded) Bioanalyzer chips. You can denature your sample (95 oC, 2 minutes followed by "snap chill" on ice) prior to loading, but the sometimes crazy results can be hard to interpret. Also, you may end up wondering whether your sample renatured, despite your efforts to prevent this from happening.

As far as fluorimetry goes, there are also single-stranded fluors, like ribo green, that can be used. But this can be a mine-field as well. The main, perhaps unexpected, issue is that ribo green fluoresces somewhat more brightly in the presence of double stranded DNA than single stranded. No where near the difference one sees with pico green, but still ~2x. We currently are trying to use ribo green, but using double-stranded DNA as our standard (phage lambda). We are denaturing our standard and our samples using the method mentioned above.

My assessment: all the library QC assays have significant issues. Beware.

--
Phillip

[HESmith]

I am wondering is any one observed that the qPCR reading is higher than the Qubit? I mean this is impossible right as the qPCR only measure the molecule which ligate with adapter.

The Qubit is specific for dsDNA. If you sample contains ssDNA with adapter ends, it will be detectable by qPCR but not Qubit. (Note: it will also form clusters, which makes qPCR more accurate for quantification.)

[niceday]

We do get qPCR results higher than Qubit and bioanalyser. I spoke to some-one at Kapa and they did say others had observed this. We use the qPCR as standard and we get reproducible results.

[Aaron Cooper]

I am wondering is any one observed that the qPCR reading is higher than the Qubit? I mean this is impossible right as the qPCR only measure the molecule which ligate with adapter.

I think if you're using the stock Illumina adapters and primers, qPCR should be accurate. It has worked very well so far for us. Are you sure you are considering the correct total size for your library and not just insert size? Also, what are you using for a qPCR standard?

I have gotten fewer spots than expected from qPCR, but this was when using a custom read 1 primer.

[anant]

I am wondering is any one observed that the qPCR reading is higher than the Qubit? I mean this is impossible right as the qPCR only measure the molecule which ligate with adapter.

[Aaron Cooper]

I think that if you have access to a qPCR machine, there really is no reason not to do qPCR.

I prefer probe-based qPCR to SYBR Green qPCR, though, since with a probe the signal is not affected by the amplicon length. This allows you to use the same standard curve for all of your libraries.