How does Illumina get to a certain final loading concentration (NovaSeq 6000)?

[GSeq94]

Hi all,

I have been very confused as to how one calculates the final loading concentration of the pool for the NovaSeq6000. I am reading the system guide for NovaSeq 6000 for denature and diluting libraries. Here Illumina specifies that starting from a library concentration of 1.5 nM a final loading of 300 pM is suggested. Now, if we assume that I am following the reccomendations for an SP/S1 flow cell:
-100 ul of library at 1.5 nM
-0.6 ul of PhiX at 2.5 nM
-25 ul of NaOH at 0.2 N (or M)
-25 ul of Tris-HCl at 400 mM.
Using the formula below
(V1M1+V2M2+V3M3)/(V1+V2+V3) there is no way I am getting the final loading concentration of 300 pM.

Can anyone explain to me how I go from these reagents at these concentrations to the final concentration of the pool that Illumina reccomends? Is that something I am missing? Can anyone explain this to me, please?

Thanks a lot and happy sequencing to all!

GSeq94

[Tom Barker]

Hi GSeq94,

I believe the final loading concentration is after the amplification mix has been added to your denatured sample which happens on the NovaSeq if you follow the standard workflow. It's not clear from the protocols but if you look at the XP loading part of the NovaSeq 6000 protocol you start with 18ul of pool, add 4ul NaOH, 5ul Tris, and then 63ul of amplification mix which gives 90ul which is a 5x dilution of your original pool. My assumption is that with standard loading it's stll a 5x dilution but only once the amplification mix is added which happens on instrument so the concentration of library that actually gets loaded on the flow cell would be 300pM in your example above even though it's still 1nM in the library tube when you load it onto the NovaSeq.