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Old 08-11-2010, 07:03 AM   #1
krobison
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Default Library quantification: opinions?

Am curious to survey how groups quantify their libraries prior to putting them on the sequencer (or sending them to a vendor, as is our case).

qPCR would generally seem to be the winner to me; very high sensitivity & accuracy and you are measuring only the active molecules

However, it seems that Bioanalyzer and NanoDrop have their adherents. BioAnalyzer does get get you some size distribution information as well, but my impression is that with low concentration libraries one is pushing the sensitivity limits of both BioAnalyzer & NanoDrop (with a resultant hit in the precision of the measurement).
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Old 08-11-2010, 08:06 PM   #2
HESmith
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You're correct that qPCR is the most sensitive and accurate. However, we've gotten fairly reproducible results using PicoGreen dye fluorimetry for quantification. We switched from Nanodrop b/c, in addition to poor sensitivity, some libraries contained contaminants that absorbed in the UV range. The dye is specific for dsDNA, and accurate for samples down to at least 0.4 ng/ul. We follow up with BioAnalyzer high-sensitivity DNA chips for size determination and to detect adapter dimer contamination (which will affect qPCR measurements). However, its concentration values can vary 2-3X from reality.
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Old 08-12-2010, 08:05 AM   #3
GW_OK
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The Nanodrop is no good for exactly the reasons HESmith gave.
The Bioanalyzer is good for sizing, but only gives ballpark quantification I've found.
Fluorometry is acceptable with some variability. I've used the Qubit system.
qPCR is the very best overall and is now what I use exclusively (with the BioA for sizing). I use the Kapa Library quantification kits on a Roche 480.
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Old 08-12-2010, 08:39 AM   #4
lee
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We use Bioanalyzer for sizing only.

Kapa qPCR for Illumina library quantification. As of August 1, Kapa kits are now compatible with the small RNA and GEX libraries. We consistently hit our target of 300-400K clusters/tile.

SOLiD qPCR assay (from Life Tech) for SOLiD library quantification.
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Old 08-12-2010, 02:44 PM   #5
VanessaS
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We also use qPCR for quantification and the Bioanalyzer for sizing. We use the nanodrop for other parts of prep.
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Old 08-13-2010, 11:56 AM   #6
Baseless
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Bioanalyzer for sizing, pico green for quantitation. We will test the qPCR once that kit arrives. I really would not miss titration if pPCR is reproducible and hits the right numbers...
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Old 08-17-2010, 07:55 AM   #7
janejane
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Quote:
Originally Posted by lee View Post
We use Bioanalyzer for sizing only.

Kapa qPCR for Illumina library quantification. As of August 1, Kapa kits are now compatible with the small RNA and GEX libraries. We consistently hit our target of 300-400K clusters/tile.

SOLiD qPCR assay (from Life Tech) for SOLiD library quantification.
I saw that Kapa also has a SOLiD library quantification kit, does anyone ever compared it with the ABI qPCR reagents?
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Old 10-07-2010, 02:59 AM   #8
suludana
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Quote:
Originally Posted by lee View Post
We use Bioanalyzer for sizing only.

Kapa qPCR for Illumina library quantification. As of August 1, Kapa kits are now compatible with the small RNA and GEX libraries. We consistently hit our target of 300-400K clusters/tile.

SOLiD qPCR assay (from Life Tech) for SOLiD library quantification.

Hi Lee,
could you please tell me your experience with the Kappa standards? I am working with Kapa kits to perform the qPCR but not with their standards.
Thanks
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Old 10-11-2010, 03:57 AM   #9
anunn
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I'm trying to quantify a library produced using the DSN normalisation protocol, which looks to be very low concentration...

Is it necessary to use the kit from Kapa Biosystems or could I use the qPCR reagents we have in the lab (SYBR GreenER from Invitrogen on an Applied Biosystems 7500)?

Many thanks,

Adam
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Old 04-08-2011, 02:18 AM   #10
ktabbada
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Our lab uses both Bioanalyzer and qPCR (the Taqman assay described in Quail et al, 2009 -- Current Protocols) for quality control and quantitation. As HESmith observed, the Bioanalyzer quantitation results are often 2-3X higher than qPCR values. I'm relieved to find that we aren't the only ones getting results of this type. However, we have occasionally had libraries where the Bioanalyzer quantitation was 10x higher than qPCR. Upon titration we found that the qPCR value gave a closer approximation of the actual concentration of cluster-forming fragments in the library.

However, I was wondering if anyone else has observed this (Bioanalyzer quantitation 10 times higher than qPCR) and if they can suggest any causes for this?

Thanks in advance,

KAT
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Old 04-08-2011, 03:06 AM   #11
TonyBrooks
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I think the accuracy is also dependent on the library prep method. Generally, the more PCR, the more accurate it's going to be to quantify by Bioanalyser/Picogreen. I think this is because amplification only occurs on those DNAs that have the correct adapter sequences ligated. Unfortunately, more PCR also means more PCR duplicates.

We mainly run target enriched samples that have undergone quite a few cycles of PCR (sometimes over 20 cycles in total) and the Bioanalyser plus Picogreen has been fairly consistent. When we run ChIPSeq and MeDipSeq we've seen more variablility, probably because the protocols used have limited PCR (4 cycles).
Running 454 genomic libraries show the most variation as there is no PCR involved, so you're at the mercy of your ligation efficiency - we would always quantify using qPCR for these libraries as the variation is way too high. For example plotting the estimated concentration of using qPCR vs Bioanalyser for our last 20 libraries gave an R^2 of 0.43, with Bioanalyser quantitation between 2 and 6 times higher than qPCR. Alternatively 454 amplicon libraries can be quantified by Bioanalyser as in theory, all molecules in the library should contain the adpaters - we found these to be consistent when using Bioanalyser.
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Old 04-14-2011, 03:46 AM   #12
Morty
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We have used the Kapa 454 kit for both Rapid libraries (gDNA & cDNA) and for amplicons for some time now, but its not working perfectly. Well, it work fine for RL, but amplicons is more of a challenge. As we are a core facility people send us amplicons produced with fusion primers, nested PCR and ligation products. Even if qPCR results and DNA chip results are in agreement, the emPCR often give very varying enrichment numbers, and we really have no good explanation for it, but we see a differense between fusion and nested generated products. Im considering skipping qPCR for amplicons and stick to chip + qubit.

Cheers!
M.
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Old 04-16-2011, 11:15 AM   #13
Aaron Cooper
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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.
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Old 07-18-2011, 07:37 AM   #14
anant
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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.
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Old 07-18-2011, 08:50 AM   #15
Aaron Cooper
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Quote:
Originally Posted by anant View Post
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.
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Old 07-18-2011, 09:09 AM   #16
niceday
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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.
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Old 07-18-2011, 09:31 AM   #17
HESmith
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Quote:
Originally Posted by anant View Post
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.)
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Old 07-20-2011, 05:12 AM   #18
pmiguel
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Quote:
Originally Posted by HESmith View Post
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
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Old 10-03-2011, 12:15 AM   #19
kulukulas
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Quote:
Originally Posted by pmiguel View Post
(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?
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Old 10-03-2011, 06:42 AM   #20
pmiguel
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Quote:
Originally Posted by kulukulas View Post
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...)

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