[GenoMax]

No I did not know that those tests even exist...

I strongly suggest not running those tests, if you are not familiar with them (and before talking with Illumina tech support).

[GenoMax]

It's not our MiSeq, we just use it from our cooperation partner. I'll check if they have a maintenance contract with Illumina. Nevertheless, I wrote tech support an email with all details.

Illumina tech support will entertain questions from end-users (so you have done the right thing) but you would need your partner to be involved if they need remote access to the sequencer to check this run/require the partner to run the diagnostic tests.

[GW_OK]

Onboard diagnostics are in the "System Check" icon under "Manage Instrument" from the main screen on the Miseq Control Software. The optics checks are fairly fast to run, the fluidics will take some time and babysitting (raising and lowering sippers as prompted).

Has your partner had any successful runs since your failed run?

Edit: Asking me to run these tests is usually what Tech Support has me do first when I call them with an issue. Enough so that I'll sometimes run them prior to calling...

[SarahAurora]

Ok that's all very good advice, thanks a lot! I hope tech support can help me!

[GenoMax]

If this failure is due to hardware/reagent problem (and the partner has a maintenance contract with Illumina) then Illumina generally will replace the reagents at no cost, so you should be able to re-run your samples for free (or at least no charge for the reagents).

[SarahAurora]

Onboard diagnostics are in the "System Check" icon under "Manage Instrument" from the main screen on the Miseq Control Software. The optics checks are fairly fast to run, the fluidics will take some time and babysitting (raising and lowering sippers as prompted).

Has your partner had any successful runs since your failed run?

No, we are the only one using the MiSeq. We already used it for 8 runs, but the ninth one was now the fatal one....

[SarahAurora]

If this failure is due to hardware/reagent problem (and the partner has a maintenance contract with Illumina) then Illumina generally will replace the reagents at no cost, so you should be able to re-run your samples for free (or at least no charge for the reagents).

That would be great!

[thermophile]

I run primarily amplicons too so the metrics that I look for are a bit different than the standards that Illumina uses (our runs don't meet the base diversity that makes some of the standard number make sense).

From the %q30 it looks like something bad happened part way through R1-amplicons aren't really long enough, optics issue, fluidics issue. Whatever it was *seems* to be machine/reagent related because it didn't improve after the turnaround. Are you spiking in primers or do your amplicons use Illumina sequencing primers? If you make an error spiking in the primers, you won't see anything after the turn around.

My list to check (roughly in order)

1. %phiX aligned. is it close to what I thought I was spiking in?
2. %base does it look appropriately horrible (my 16s amplicons should be at 90% for the first ~50 bases, then decline to ~80% for the rest of the read)
3. Cluster density across flowcell, top and bottom
4. thumbnails, does the density seem about right
5. fwmh. The software is not made for 90% single base, focus issues are common, but fwmh shouldn't be too jagged