I was provided with a protocol a few years ago by illumina to use when sequencing really weak libraries on the MiSeq, when a large volume of library was required, and I just carried on using it.

The protocol used the same 0.2N Tris-HCl pH7 that the NextSeq protocol uses but instead of adding the same volume of Tris as you do of NaOH you add a volume equivalent to the volume of library and NaOH, I guess this got tweaked and the result is what we see in the NextSeq protocol.

Knowing that this works with no problems I am sure the NextSeq protocol would work fine on the MiSeq.

Kevin, just to clarify: if you denature 40ul of library with 40ul of NaOH, you neutralize with 80ul of 0.2N Tris-HCl?

I'm particularly curious, because I just attempted using the current(?) NextSeq protocol, neutralizing with the same amount of Tris-HCl as NaOH, and I ended up with low cluster densities. (~180k/mm^2) My suspicion was that I hadn't fully neutralized my library, and that there were still OH ions preventing clustering on the flow cell...

Does anyone know if there's a downside to adding a little extra Tris-HCl?

In that case, following the protocol I was sent, you would add 80ul of 0.2N Tris.

What type of libraries are you loading?

I loaded 16s rRNA amplicon libraries, V3-V4 region, ~464 bp insert length.

I will say, I'm not 100% certain I should have trusted the stock of Tris-HCl that I used... but I wonder if it would have worked had I used the protocol that you were sent. I get the impression that even a little bit of extra NaOH has an effect on cluster density, and that it might be better to add too much Tris-HCl than not enough...!

Depending on what method these libraries were produced with I might have had a similar problem in the past.

We were using the method from the Earth Microbiome Project doing a one step PCR to amplify, add adapter and indexes. Concentrations looked good, fragment size was good and the PCR products were clean but cluster density was really low.

We were using 600 cycle V3 kits and in the end I just kept increasing the loading concentration, while using the Tris neutralisation.

Have you also been doing the heat denaturation outlined in the Illumina 16s Metagenomic Library prep protocol?

Dear Kevin. Thank you very much for your answer. I have tried it without Tris-HCl and got satisfactory results. But I think adding Tris-HCl is a good idea. I wonder why Illumina doesn't recommend it?

Dear Kevin,

The trick looks very nice. However a silly question, do you think excess HCL can adversely affect the clustering in MiSEQ