No clustering

[RedBix]

Hi all,
I'm having troubles with a MiSeq run. I used a Multiplicom NGS kit, specific for two genes. I followed the protocol and produced 4nM libraries. I pooled these libraries and than denaturated the pool (5ul pool + 5ul NaOH 0.2N fresh made) for 5 minute. I added HT1 buffer to 20 pM concetrantion, than diluted again to 12 pM and loaded this pooled diluted library togheter with 5% PhiX equimolar. I loaded the 3 custom primers requested by the protocol (read1, read2 and index) at the appropriate concentration. I started the run with correct sample sheet but it stopped before the first cycle, it seems for the impossibility to form clusters. The NGS kit I used was previously used in the lab without problems and I've tried to quantify libraries with tapestastion and the images I see are in agreement with the correct lenght of my libraries. I can't understand what went wrong and I'm wondering if can be a reagent issue. In few days I will reload the libraries, so a would like to understand why the instrument (MiSeq) stopped so early.

Could someone tell me his experience) Thank you in advance

[helskel]

Possible Sample Sheet issues

It could potentially be an issue with your sample sheet. Did you use Illumina Experiment Manager to make your sample sheet? Also, did you spike your custom primers into the primer locations, or move it to the custom primer locations?

If you selected for custom primers, it should have C1, C2, C3 on your sample sheet. In that case, you must use the custom primer locations on the reagent cartridge.

[kmcarr]

This is not directly in answer to your question, but is something you need to think about if you are using custom primers and still adding PhiX to your run. If you put the custom primers in the designated wells of the reagent cartridge, and then edit your Sample Sheet so that the MiSeq will take the custom primers from those wells, the MiSeq will NOT use the standard primers which are pre-loaded in the reagent cartridge. This means that there will be no primers to sequence your PhiX control DNA clusters.

When our lab has to add custom primers to a MiSeq run we do not use those custom primer wells set aside in the reagent cartridge. We mix the custom primers into the appropriate wells with the standard R1, R2 & I1 primers (don't ask me which numbers those are because I can't remember off the top of my head). There is no editing required for the sample sheet. The MiSeq will use the default primer wells which will now have both your custom primers and the standard ones needed for the PhiX control library.