[Nitrogen-DNE-sulfer]

Any tricks for avoiding the massive library dilutions steps on the MiSeq. The neutralization seems like an excessive dilution which could be done with more concentrated HCl? We're having to amplify 5-10 more cycles to get Amplicons to the right concentration so we can survive these enormous dilutions.

[pmiguel]

The MiSeq wants 600 ul loaded in the reagent cassette port. So, I guess you are stuck with that. The denaturation using HT1 is 50x to get the base concentration within specifications. But I guess it is the total volume that is the issue, not the increase in volume resulting from neutralization.

--
Phillip

[ECO]

Any tricks for avoiding the massive library dilutions steps on the MiSeq. The neutralization seems like an excessive dilution which could be done with more concentrated HCl? We're having to amplify 5-10 more cycles to get Amplicons to the right concentration so we can survive these enormous dilutions.

We've totally stopped using ILMNs protocol because frankly it's ridiculous. We denature with 2N NaOH (to 50mM final), then neutralize with 2N HCl. Then you can dilute at will as long as your final HT1 isn't diluted more than ~10%.

To get around this...I've successfully done the denaturing in 300ul (for multiplexing 48 dilute libraries), then instead of using HT1/"hyb buffer" to dilute, used 20xSSC and 10% Tween20 to adjust the sample to the final template, salt and detergent concentrations for efficient hyb. (which are 5x SSC and 0.1% T20 when it hits the flow cell). We've also added TrisHCl to 5-10mM as described in the "Sanger improvements" paper with little effect, but it's probably good for robustness.

Good luck!

[ECO]

Oh and yes, I am slightly worried about library depurination from the HCl but in practice we haven't seen any differences in error profiles or yields...