MiSeq run with low Q30 and high cluster PF

[haroldnunez]

Hi all,

Could anybody give me an idea or though on the following?

I made a run with the MiSeq using the TrueSeq LT kit and 151 cycles. It was an amplicon library with 3 different DNA, everyone with his own index. The run has very low Q30, around 6%, but has Clusters PF (passing filter) of 85%. The number of clusters is around 1100 K/mm2. Moreover, no reads were identified.

Could have been the PhiX? it was set at 15% but not freshly made, only two weeks old (protocol recommendation is 3 weeks max).

Anyway, any help will be greatly appreciated. Bye.

[GenoMax]

You should contact Illumina tech support. They should be able to do a remote login and look at this run. That may be the fastest way to diagnose what is going on with this run.

That said: What do you mean by "no reads were identified (no sequence or did the samples not demultiplex)" when you had 85% clusters that passed filter (leaving the Q30 aside for now since this was an amplicon run).

[haroldnunez]

Originally Posted by GenoMax

That said: What do you mean by "no reads were identified (no sequence or did the samples not demultiplex)" when you had 85% clusters that passed filter (leaving the Q30 aside for now since this was an amplicon run).

Thanks for your advice. About your comment, it should have said no Index were identified. I have 18,096,508 reads but none is assigned to an index.

Anyone has experienced something like this before?

[GenoMax]

If you are sure that the index read has worked and for some reason the demultiplexing did not then you can:

a) Try to re-run the analysis with MiSeq reporter or if that does not work
b) try doing the basecalling/demultiplexing using bcltofastq on a separate unix server (for ref: http://seqanswers.com/forums/showthread.php?t=39153)

In any case first see what Tech Support says (I see that you are in Chile but hopefully there is a way to contact the tech support either local or the one in US).

[haroldnunez]

Originally Posted by GenoMax

If you are sure that the index read has worked and for some reason the demultiplexing did not then you can:

a) Try to re-run the analysis with MiSeq reporter or if that does not work
b) try doing the basecalling/demultiplexing using bcltofastq on a separate unix server (for ref: http://seqanswers.com/forums/showthread.php?t=39153)

In any case first see what Tech Support says (I see that you are in Chile but hopefully there is a way to contact the tech support either local or the one in US).

I will try your suggestion, however I'm worried about the quality score. It could improve if I do the basecalling/demultiplexing with bcltofastq? or I just will recover the index?

[GenoMax]

Was this a reagent V2 or V3 run? 1100 clusters/mm^2 may be bad news for the tag read if these are V2 reagents. You may have to repeat the run in that case. Tech support can determine if the run was overloaded by looking at the thumbnails.

[haroldnunez]

Originally Posted by GenoMax

Was this a reagent V2 or V3 run? 1100 clusters/mm^2 may be bad news for the tag read if these are V2 reagents. You may have to repeat the run in that case. Tech support can determine if the run was overloaded by looking at the thumbnails.

Bad news indeed, it was a V2 run. Some thumbnails.

s_1_1111_a.jpg

s_1_1111_c.jpg

s_1_1111_g.jpg

s_1_1111_t.jpg

[GenoMax]

You can push cluster numbers beyond the recommended limits (http://www.illumina.com/systems/mise...fications.ilmn) for normal sequencing but in case of multiplexing one runs the risk on losing the run (like in this case, even though actual sequence reads are probably acceptable).

If these are amplicons you should stay within (lower end of) the recommended limits (800-900 clusters/mm^2 for V2).

[ECO]

Look at the base diversity of read 1.

[haroldnunez]

Originally Posted by ECO

Look at the base diversity of read 1.

Is way off to A and T. I should repeat it, however it is wise tu use the same library?

Could PhiX be the problem as stated before?

[GenoMax]

Can you post a FastQC plot of the distribution?

Irrespective of not being able to demultiplex the data does the sequence you have look to be the right mix of what you expect? If it is not then you should think twice about resequencing the same library.

[Shimul]

Hi There,

I am running my bacterial sample using MiSeq V3. After 70 of 600 cycles, it showed cluster density is 1092K/mm, cluster passing filter 91.4% and the >=Q30 is 96%. Do you think that the run is going well?

[GenoMax]

Originally Posted by Shimul

Hi There,

I am running my bacterial sample using MiSeq V3. After 70 of 600 cycles, it showed cluster density is 1092K/mm, cluster passing filter 91.4% and the >=Q30 is 96%. Do you think that the run is going well?

So far yes.