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
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
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