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mkalive73 09-27-2013 12:54 PM

using NEB kit to make libraries for Illumina MiSeq
 
Hello,

I used NEB multiplex kit to make libraries for Illumina MiSeq. Trying to figure out the amount of sample to load in the flow cell to get a decent cluster density. Last round I loaded 12pM of total sample and it over clustered with my coverage very low. My colleague uses 7pM of the same kind of sample on a HiSeq, how does that compare with MiSeq? should Miseq sample amount be lesser or higher?

Thanks for your suggestions.

Isequencestuff 09-30-2013 07:54 AM

what MiSeq kit (flowcell) are you using? I assume you're using the V2 kit since you're aiming to load 12 pM of total sample. If your quantitation method is accurate, aiming to load 12 pM should give you a perfect cluster density. We usually get around 850-900. Our quantification process: qPCR post library construction, then based on those quantities dilute all library samples to 2 nM and pool. Then run a last qPCR on that one pooled library sample to make sure it's close to 2 nM (this saves us from wasting a flow cell). From there we dilute to 12 pM final (including about 1-5% phiX), then load and go.

MWN 09-30-2013 09:58 PM

For V2, I load 11 pM based on Qubit. For V3, 14 pM.

kcchan 10-01-2013 10:13 AM

Comparing loading concentrations isn't very beneficial. Long inserts also don't behave in the same way as short inserts, so a 12pM TruSeq DNA library may be fine whereas a 12pM miRNA library will overcluster. Also, each instrument has its own different sweet spot. The same sample at 12pM may be perfect on one MiSeq but overcluster on a different MiSeq. It's better being more conservative (4-6pM) and working your way up over the course of a few runs.


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