[Genquest]

Hi HESmith,

Thanks that you took time out to reply!

That's what I would have expected when I repeated the qPCR for same libraries after two months.But I got alarmed,when I saw that my cDNA libraries increased in concentration (just double) in comparison to the previous qPCR done.
That is why I reached Seqanswers because cannot think of any possible reasons or explanations.
Can anyone give their opinions and insights on this?

[HESmith]

We have the occasional request for sample resequencing, and often see a significant decrease in library concentration upon requantification. Nucleic acids adhere to plastic during freezing (a well-known phenomenon), thus the recommendation for using Lo-Bind or similar tubes. The effect is negligible at high concentration, but a real problem at the concentrations typical of sequencing libraries.

[Genquest]

Thanks Luc,

Waiting for others to respond as well?

Do any one get request on re sequencing same samples from their clients?Did anyone redo qPCR's on cDNA libraries to check its integrity over a period of few months?

[luc]

No.
We, are not re-doing the qPCRs and have never seen loading problems.

[Genquest]

Hi all,

Back again after three years of peaceHope people are still responding to the queries here.We have switched completely to Kapa Standards now.Assays working completely in sync except for few variability here n there.A quick question….

Have anyone of you experienced a variation in qPCR of cDNA Illumina libraries?

Has anyone seen a difference of two times in qPCR concentrations,when re quantitated after freezing those libraries for two months and then compared with its own qPCR concentration,which was done at the time when the libraries were made?

Thanks

[Sciurus]

Okay, thanks!
Our technician suggested running a NuSieve gel and re-purifying with beads.
I'm not entirely sure if that will help any but I'll probably try and then re-do the libraries if that doesn't help.

[pmiguel]

Tony already covered the main point. Lesser point, the secondary bulge at a higher molecular weight likely comprises bubble products. But these are also only really an issue for accurate titration of samples prior to clustering.

--
Phillip

[TonyBrooks]

We saw this before with the in line controls

http://seqanswers.com/forums/showthread.php?t=12543

The library will sequence OK as is. Quantification may be more of an issue as it's harder to pick the correct size to normalise to. If you want to improve the library and get better quantification, you'll need to reprep it. You can't fragment it now as the adapters have already been ligated.

[Sciurus]

That's your library. It looks like you slightly under fragmented. Maybe next time increase length of the fragmentation incubation. We saw this with our NEB Ultra RNA prep's. It still sequenced OK, but it's slightly harder to quantify.
Not too sure about the shaper HWM peaks. Did you use any in-line controls?

Thank you!

So, this means that I would have to do a whole new library prep or can I re-fragment these samples?

I used the in-line controls for end-repair, a-tailing and ligation but as far as I understood, I can only check if they are present after sequencing?

[TonyBrooks]

That's your library. It looks like you slightly under fragmented. Maybe next time increase length of the fragmentation incubation. We saw this with our NEB Ultra RNA prep's. It still sequenced OK, but it's slightly harder to quantify.
Not too sure about the shaper HWM peaks. Did you use any in-line controls?

[Sciurus]

Maybe here somebody has seen something similar like my results from the BioAnalyzer run following RNA library prep (and pcr) and can tell me where the huge peak comes from and how to proceed now?

[Genquest]

Thank you all!! I was bit hesitant to post,until no option was left with me..
Now I feel that lot many queries can be resolved by open discussions.

About my library type..it has been genomic and metagenomic!!
The one that generated 20% less cluster when quantitated with qPCR was metagenomic library!!I agree with Phillip...multiple reasons prop up while troubleshooting!!
Conclusions were drawn that qPCR were set up incorrectly giving faulty concentration(R^2 values were 0.998 &E=85%).Later I came to know that the concentration that was taken for clustering was presumed by GA results and also a Hiseq Run(in which qPCR was not run for QC) but had given optimum cluster nos. that had been shooted for!!

The second set of libraries were from some client,so don't know about the sample type.
This behaved so weirdly!!Our Kapa sybr fast was over so decided to use ABI Sybr fast for qPCR.The concentrations were very high..so we repeated to check out for handling errors!!But second time concentration turned out to be even higher....got trapped in endless confusions.Then borrowed a Kapa Sybr fast to repeat the same libraries and all concentrations fell in place.Then concluded that ABi sybr fast was behaving differently with Illumina libraries so now we have stuck to Kapa!!

Now with all these discussions can we draw a conclusion??Can anyone summarize the do's and don'ts for qPCR quantitation and what else should be taken care of to get accurate results??

Also one more question is there any data available for cluster generated for PhiX standard dilution?Thanks

[pmiguel]

Looks like quite a few discussions of this issue throughout the forum today. Seems to me that we want to distinguish between "major" and "minor" failures to correctly titrate a library. The cut-off between the two would be arbitrary. But I would say anything less than a 2x difference in what you thought the cluster density would be and what it actually would be reasonable.

If you are consistently getting within 2-fold of where you thought you were, you are in a completely different situation than if you are (for example) 9x wrong. Less than 2x suggest that your assays need a little tweaking. Maybe check to see if you just have a systematic bias that can be corrected using a static factor. Or if things are random, then looking for ways to reduce the noise in your system are called for.

Whereas if you are >10x off then you are looking more towards catastrophic issues.

Even though I frequently rail against people not distinguishing between an assay being 30% off or 300% off, I was recent bitten by failing to do exactly this. I had a flow cell badly over cluster. In the throws of grief and horror, I remembered that I had failed to correct for the longer length of my libraries with respect to the standard. This came to as much as a 1.5x correction factor.

We re-clustered using the new factor. Still over-clustered! Turns out there were a host of other issues with our estimations, the greatest of which was that our phiX standard concentration was wrong!

Anyway I have seen a "when it rains, it pours" phenomenon occur on more than one occasion. That is, something that used to work, no longer does. But when one looks into it, there was not one point of failure but a whole host of them. I think this derives from an attempt to keep our methods agile and costs low -- most standard QC is discarded. But also, it deals with the robustness of the method itself. If a set of methodology is fairly robust, no single minor issue is likely to produce systemic failures. So by the time you are troubleshooting, you are not tweaking or correcting a minor issue, you are dealing with a multitude of issues.

--
Phillip

[sehrrot]

One more quick post,

A bit different behaviour between GAII and Hiseq in terms of finding the cluster density and actual showing on the cluster, me and my colleague think..

[sehrrot]

hi all

1. Kapa kit is the best option (at least to me and my colleagues). (to Tony) please check your Kapa kit's standard, then you will get nearly 100% curve (as I always did).
2. optimising the optimal cluster is a sort of relative thing. Once you have training run, you will need to run PhiX to find a optimal range of the cluster density (recommend to try few pM ranges): so I cannot say the absolute value of this.
3. what I concern for Genquest's question is,,, what was you library type? whole genome? exome? RNAseq? My feeling is that,, ,whenever I did RNAseq, its qPCR always gave a variable range of actual molarity. For example, I measured an RNAseq sample as 10nM but it came as 90nM. I am pretty sure this variable would be attributed to variable length of library, which is also involved in calculating the molarity. So, for RNAseq library, I really recommend to measure the "exact size" of the library
3-1. For RNAseq library, you should be careful about contamination of probes or primer dimers. Even if you see very tiny portion of this in bioanalyzer peak, those will cause serious problem in sequencing, I mean "decreasing library complexity". Of course, I pretty assume that those tiny fragments will affect on the molarity of a library.
3-2. Regarding whole genome & exome library, you wouldn't be worried about qPCR things. If you still get bizarre result in qPCR, you will need to check either your pico-green or qPCR method.
4. I haven't used Illumina qPCR protocol for long time since I got Kapa kit. It's bit sad that Illumina qPCR protocol has the weird description for serial dilution of a library (it might be corrected now though..)