Next-seq starting library concentration for library denaturation

[Irene]

I am following the Nextseq System-denature and dilute libraries guide. The lowest starting library concentration recommended for denature libraries is 0.5 nM. However, the conc. of one of my libraries is particularly low and the final volume would be more than 40 ul.

I would like to know if the volume of the denaturation step is important. If I dilute my libraries at the concentration of 0.25 nM in 80 ul and add 80 ul of 0.2N NaOH followed by 200nM Tris-HCl pH7.0. Would this also work?

[huguesparri]

Hi Irene,
First of all, I don't have experiment with the nextseq system. We have a Hiseq2500 but I suppose the issue and solutions are the same.
What we usually do when our library concentration is too low is to add a neutralization step after the NaOH denaturation: we add 0.1N HCl to neutralize the NaOH.
The point is to be sure that we don't end up with a NaOH concentration higher than 0.001 N(1mM) in your final library dilution (the one you're going to cluterise with). Failing to do so could inhibit the library hybridizaiton on your flow cell and give you a lower cluster density.

[Irene]

Hi huguesparri,
Thanks for the tip.