Illumina MiSeq High cluster density, but low %PF problem

[setramis]

Hello everyone.

Please, I need a help with my MiSeq genome sequencing run. I am sequencing M. tuberculosis genomes using Nextera XT and a 500 cycles v2 cartridge/flow cell. I always obtained aproximately 950-1050 K/mm2 and a passing filter more than 85%; However, in my last run I obtained 1063k/mm2 (i think it is ok), BUT JUST 50,3% %PF... sincerely, I dont have any idea what happened. I know with overclustering there will be a lower %PF, but in think is not my case. I s the first time i have this issue.

Thank in advance for your answers.

[finswimmer]

Hello,

take a look at the cluster pictures. If you overcluster to much, the MiSeq cannot detect all clusters correctly and reports a much smaller number.

fin swimmer

[GenoMax]

1000K/mm2 is outside limit per sepc for V2 chemistry. While it is tempting to push cluster density (trying to get additional data) the fall in data yield/quality is precipitous when you go over a certain density (like you discovered). You would want to carefully look at the data you have to make sure there are no artifacts.

[provirus]

Have a look at your intensities of the first 25 cycles (e.g. in the sequence analysis viewer) (which might not only be a problem for amplicon sequencing). Below a quote from a Illumina white paper:

The %PF calculations involve the application of a chastity filter to
each cluster. Chastity is defined as the ratio of the brightest base
intensity divided by the sum of the brightest and second brightest
base intensities. Clusters “pass filter” if no more than 1 base call has
a chastity value below 0.6 in the first 25 cycles.

[setramis]

Thanks for your answers ... I read all of it., I got surprised because at two previous runs the last year I got a cluster denstiy of 1200 and 1100k / mm2 aproximately (definetly an overclustering) but the passing filter was approx 80%. Then I recalculated my steps and I corrected that. But now, I think I did not get it too much (just 1069K/mm2), and the passing filter fell too much.

Now I am suspecting for the machine, MISeq. Because other people used to have the same, even worse, results with passing filter fell until 15%.

Now I taked a look for my intensities at the first 25 cycles (like "provirus" recomendation) and I notice some extrange: first, definetly i got overclustering; second, the "T" base shows a marked overclustering (see picture), and thats the same result for all the run. could there be a particular reason for such behavior?



For now, given the picture intensities Will it decrease my input DNA from 20 ul to 18 ul?

Thanks for your answers and time.

[setramis]

whats i hapennig... all my replies never come out

[GenoMax]

whats i hapennig... all my replies never come out

It looks like your replies were caught in the moderation queue. I just approved them.

It appears that you did not attach any images though (if you thought you had). You can edit your post and attach them.

[V_Ang]

Hello everyone

We are currently experiencing a sequencing issue with our Miseq libraries (made with DNA prep Illumina kit). For several runs now, we've been getting very low % PF clusters (<to 5%) with a high cluster density limit (1000-1100 k/mm2). We think it's overclustering, by rerunning more diluted, we get better results but we don't understand where this overclustering comes from. We had 3 out of our 4 last runs which were overclustered. Our bioanalyzer profiles are ok, and our concentrations dosed to both Qubit and Tapestation are in the usual ranges. We've checked all our dilutions, which are OK too.

I came across an old post by Setramis (quoted above) that talked about this same problem and looking at the images, I have the same observation: an overclustering of T and a %base in the SAV that indeed shows a very strong overrepresentation of T.
Does anyone have any idea about the cause of this problem?

Thank you very much for your help.

Thanks for your answers ... I read all of it., I got surprised because at two previous runs the last year I got a cluster denstiy of 1200 and 1100k / mm2 aproximately (definetly an overclustering) but the passing filter was approx 80%. Then I recalculated my steps and I corrected that. But now, I think I did not get it too much (just 1069K/mm2), and the passing filter fell too much.

Now I am suspecting for the machine, MISeq. Because other people used to have the same, even worse, results with passing filter fell until 15%.

Now I taked a look for my intensities at the first 25 cycles (like "provirus" recomendation) and I notice some extrange: first, definetly i got overclustering; second, the "T" base shows a marked overclustering (see picture), and thats the same result for all the run. could there be a particular reason for such behavior?



For now, given the picture intensities Will it decrease my input DNA from 20 ul to 18 ul?

Thanks for your answers and time.