MiSeq error: Through-focus peak did not exceed SNR threshold

[user 31888]

Hi,

Using a 10 pM pooled library of 6 indexed samples (4 from FFPE + 2 from fresh tissues) prepared with TruSeq Exome kit, I got this error message when running on MiSeq with no data generated (except the log files):
'Through-focus peak did not exceed SNR threshold (Illumina.Instrument.Bolt.Logic.AutoFocus.DarkSampleException)'

Do you have any idea what it means?
What could be the cause of this error?

Thanks !

[GenoMax]

That probably means that no signal was detected (no clusters generated). Was the pool QC'ed?

You should contact Illumina tech support and have them take a look at the run. If this is a hardware/reagent failure, they will be able to determine and replace as needed. Include a screenshot of the error on MiSeq with your ticket.

[user 31888]

Ok.
I checked the concentration of the final library with PicoGreen.

How do you QC the final pooled library?
Any method/kit in particular?

[lxkx]

We use the qPCR based method for quantification, such as the KAPA kit.

[Jessica_L]

To expand on lxkx's answer:

One of the issues with PicoGreen is that it only looks for dsDNA, which you might have plenty of, but ignores the potential complication that not all DNA fragments will have sequencing adapters in the correct orientation. qPCR amplification only occurs when adapters are in the correct orientation, so it's a more accurate method when quantifying material for sequencing. I've used the KAPA kits for several years now and have had very good luck with them. The standards are remarkably consistent lot-to-lot, too.