Discordance between Cluster Density and Thumbnail images

[soleulloa]

Hi all,

I ran MiSeq using a 500 cycles kit.
The value for cluster density was 310K/mm2 but if I'm going to look the thumbnail images, I think I have much more cluster than that.

Illumina tech looked the results and says "you are overclusterized".
But for me cluster density is OK when I've reviewed the images.

I'm attaching here 4 images from cycle 1.

Could you tell me your opinion?
Thank you!!
Soledad

[nucacidhunter]

To me reported cluster density is what I would expect from the images. The run has been underclustered for a library that looks high diversity although I am assuming that the base diversity of other cycles are similar to the first cycle.

[Brian Bushnell]

What's the scale on the little yellow boxes?

[misterc]

That looks about right to me for 300-500K/mm2 raw density (and NOT overclustered). Those intensities look reasonable to me too for cycle 1 given the MiSeq ramps up exposure on the cameras at each cycle to counter the typical decreasing intensity plots on their instruments.

[soleulloa]

Ok! Thank you.
I'm using 10 pM (from 4 nM) to get this result.
What do you think I have to use now? 12 or 15 pM to get a good run?

[microgirl123]

What's your percentage passing filter?

[soleulloa]

Hi,

95.27% PF

[microgirl123]

If 95% of them are passing filter, then you are definitely not overclustered. If you were overclustered your percentage passing filter would be low.

Unfortunately, the relationship between loading concentration and clusters is not linear so it's always a guess. I find that getting good cluster density is the Achilles' heel of the Illumina Miseq. My guess would be to go with the 15 pM since your density was so low.

[pmiguel]

What's the scale on the little yellow boxes?

I don't know, but when I looked into it in the past, it seemed that the size of a cluster was around 1um in diameter. But that may have been on a HiSeq. I'm not sure whether the scales would be different on a MiSeq.

Old-timers like @ECO would be more likely to know. (Alas, I don't think @ callouts actually work on SeqAnswers...)

--
Phillip

[GenoMax]

Illumina has a note on optimizing cluster density available here.

@soledad: Have you checked what the images look like in the middle and at the end of run?

[soleulloa]

Someone has quantified libraries before dilution? When you prepare 2 nM dilutions?
I've quantified before and after that final dilution now because this is the first run when I have this problem and I'd like to compare these results.
Always I was between 1000-1200 K/mm2 using a 300 cycles kit.
This time I did the same process... and the only variable is I'm using 500 cycles kit.
But the library concentration will be the same.. so I don't understand what happens.

[nucacidhunter]

It is most likely overestimating library concentration if you have used the same type of library. qPCR is the best method for quantification, other methods that quantify dsDNA are inadequate for libraries that contain non-sequenceable dsDNA fragments.