[EmiVig]

Hi pmiguel,

thank you very much for your hilightening reply and here I am for an updating!
As suggested, I measured my histone ChIP-seq libraries using the KAPA kit (I used the DNA standards provided by the kit and I followed the instructions for size-adjustement) and I ended up with almost double the concentrations than the Qubit, therefore this would sustain the hypothesis of flowcells overloading. Without size-adjustment instead, the concentration resulted instead lower. So my first question is: is it right to perform a size adjustment? I would do it, since indeed my libraries have a different average size then the DNA standards.
At the same time, I've generated a standard curve using PhiX, as suggested by Illumina, because our sequencing facility tested the cluster generation using PhiX. I obtained a lower concentration (without size-adjustment even lower...) than what I obtained initially with the Qubit!!
At the end, I got back two opposite results ...I'm a bit stuck now...I would be more prone to believe to the high concentrations measured by KAPA qPCR (using KAPA DNA standards and size-adjustemnt), since I might have a good portion of "ssDNA bubble product" which the Qubit is not able to detect ...but how could I be sure of that? I'm thinking of sequencing in parallel the samples taking in consideration one or the other concentrations...but it would be a waste of material/time/money..
Moreover, last time I multiplexed 3 samples per lane...might also the low-complexity being an issue? I would like to barcode 6 samples per lane this time.
Please, find attached a summary of the concentration values obtained with the different methods + I also attached the Sequenicng Analyzer Viewer of my failed sequencing (lane 7 and 8). I'll attached as soon as possible the Bioanalyzer traces of the libraries.
I hope I'm not bothering too much, but I think this might also be helpful to somebody else encountering similar problems...
I would really appreciate any help! Thank you so much in advance!

Emilia

[pmiguel]

Hi Emilia,
With the additional information you provide I'm 90% certain that your samples were overclustered. My advice is to trust the KAPA results an size adjust based on the size of the shorter-length peak.
If you want to know whether high bias is an issue, then set your SAV "data by cycle" pane to "% base" and it will be immediately apparent.

--
Phillip

[pmiguel]

By the way, don't trust the concentration of Illumina phiX, it is always much below the 10nM it is supposed to be. The KAPA standards work fine.

--
Phillip

[aelhage]

Dear all
I do have double peaks in my dsDNA HIGH SENSITIVITY bioanalyser traces of rna-seq libraries (prepared using NEBNext ultradirectional RNA library prep for illumina cat. no. E7420). The smallest peak around 250-300bp and the largest one around 1500bp.
I can understand from your very interesting posts that I should send these libraries for illumina sequencing providing accurate measurements of DNA concentrations would be performed by KAPA q-PCR kits? I hope I did get this right?
Alternatively, I could add extra primers and perform a second standard PCR on my library with only 1 cycle with a 2 min denaturation step, hoping that the extra peak would disappear!?
Thanking you very much for the great advice you are providing!
Best wishes
AEH

[AmitChaurasia]

I don't think there is much chance that "AndyG" is still monitoring this thread, but just in case anyone reading this now would be helped:

Those double peaks are almost certainly a main peak and a "bubble product" peak. AndyG's protestations to the contrary suggest he doesn't get the concept.

Bubble products are said to form when adapters from 2 product strands anneal to each other. Because a great number of different inserts will comprise a library, it is unlikely that any 2 product strands will be complementary in the middle. So you end up with the "bubble product", a molecule annealed and double stranded at either end but the non-complementary middle section remains as a double single-strand "bubble" domain.

As you might imagine, this floppy thing migrates more slowly than its more compact fully double-stranded brethren. An Agilent chip will not correctly estimate its molecular weight by a variable amount. The fact that the two peaks overlap one another is not a sign that one is not a bubble peak.

Normally bubble peaks cause no issues for sequencing as long as you use qPCR to estimate the concentration of your libraries.

But, if you see bubble peaks, it is an indication that your PCR primer concentration has become limiting in the PCR reaction. At high primer concentrations, the primer annealing to a product strand will be kinetically favored over two product strands annealing.

Strand melting your library and allowing the strands to find their correct complements is a waste of time. Don't do that.

If you can't stand the idea of bubble products in you sample, you could probable get rid of most of them by adding your library to a fresh PCR reaction, with lots of primer and then doing a single cycle -- starting with a denaturation. This would allow the high amounts of primers to anneal and be extended into a double-stranded product.

But this is also a waste of time. Again, the bubble products don't actually cause problems.

They are an indication that you used more PCR cycles than were necessary. So you could cut back the number of cycles next time you made libraries.

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Phillip

This is truly helpful to solve my long pending case with the customer...

Kindest Regard's
Amit

[pmiguel]

This is truly helpful to solve my long pending case with the customer...

Kindest Regard's
Amit

Glad to help...
--
Phillip