16s updated NextSeq500 library prep for low concentration ok for MiSeq ?

[BioGenomics]

Hi all, anybody has experience with using a 0.5 nM 16s library concentration (instead of 2nM) on the MiSeq ? The NextSeq500 has this protocol (updated October 2014, so more recent) and we thought to add the 200mM TRis buffer PH 7.0 too to hydrolyse the higher NaOH concentration needed initially. Thanks

[nucacidhunter]

Hi BioGenomics

Would you post the result if you could. In theory it sounds fine, but one always wonders why Illumina is not recommending it for their other systems.

[BioGenomics]

Hi NAH, Illumina TechSupp replied as such on the use of NextSeq500 protocol:

"All of Illumina platforms use isothermal amplification for clustering, and sequencing by synthesis for sequencing. So the diluting and denaturing process is similar for all platforms; with the exception of volumes and concentrations, that should be modified to meet the optimal cluster density and data yields as per the specifications of your instrument of choice.

So, yes you can use the same low concentration diluting and denaturing protocol for the MiSeq keeping in mind that the loading volumes in lower (600ul vs 1300ul), and your loading concentration should be higher (6-20pM vs 1.8pM).

For more information please see:
Preparing Libraries for Sequencing on the MiSeq http://support.illumina.com/content/...15039740-d.pdf

Denaturing and Diluting Libraries for the NextSeq 500 http://support.illumina.com/content/...-15048776d.pdf
"

So we made a 0.5 nM library now, used the 200mM Tris-HCL pH 7.0 additional step to neutralize the increased NaOH concentration and loaded all at 12 pM end concentration today (we tested 4 and 8 pM already with a bit too low clustering, 5 % PhiX) . First results 800K Clusters, so looking good ! Now waiting for the Q30 % in the next days. Illumina should update more regularly their protocols...

To be continued...

[nucacidhunter]

Thanks BioGenomics for posting this. I wonder if you are using v2 or v3 and what type of library you are sequencing.

[BioGenomics]

Using v3 and running pool of 16s amplicon libs + Nextera XT small genome/large amplicon libs. So the 5 % PhiX might not be needed. Run still looking good (98% > Q30).