I have run some chips (318) on Ion Torrent but I am getting high polyclonal beads many times. I have tried reducing my library concentration but it has not worked for me, can anybody suggest a checkpoint to look for my error? I think the bioanalyzer profile may be faulty, I have tried a few different concentrations but nothing seems to be working.
How much DNA are you using? Silly question but have you double checked your library dilution calculations?
I startd with 100ng DNA for library prep. yeah I have checked and rechecked my calculation... what i think the problem may lie with overestimation of bioanalyser...
I have been using our multina (same as bioanalyser) to determine size, but using the qubit to determine concentration, and then use both size and conc to determine dilution. It has been working really well with this method.
Perhaps you can make serial dilutions of the starting material for the library and run qRT PCR on a housekeeping gene or gene of interes for each of them. This may give you a better idea how accurate are your analyzer estimates.
If you do qPCR, which is considered gold standard for the Illumina systems, you should quantify your whole library, and not a housekeeping gene, using primers specific for your adapters. You need a standard with known quantity (number of DNA molecules) for absolute quantification. For Illumina there are kits available, I think for Ion Torrent that should be the case, too...
I found this after 2 min on the Life website: http://products.invitrogen.com/ivgn/product/4468802
it's a qPCR kit with standards, costs about 1k €.
One possible reason for polyclonal ISPs is excess of library over beads. Are your beads ok? If a ~10-fold dilution of library is the same and your dilution calculation is ok then this can be excluded. Another possibility is a poor emulsion. Then, multiple beads and library molecules would crowd a water droplet.
Karol and Stephan, I tried the ISP Quality control kit for ISP quantification after dilution and proceeding with bioanalyzer results. I compared my results with what others get on Ion Community, and my unenriched beads are crossing the 100% percent as compared to 30-50% recommended by them. My enriched beads are also crossing the 100% as required but i cannot account for the high percent of unenriched beads. I have obtained proper results in my Ion Torrent in the past but this problem is coming in last 2 or 3 samples i tried running.
Karol, I have One Touch installed and it takes care of the emulsion steps. I think i should not worry about the One touch procedure. May be i am giving wrong input to it.
Another thought i am having is that may be my chemicals are not in good condition.
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