MiSeq run_Error

[Steffi1]

Hello,

Can anyone give me any help on this problem: Today I started an amplicon sequencing run on the MiSeq platform and after hundred minutes a window popped up with the following error message: "Poor-quality focus model data" Aborting run!

Thanks a lot!
Steffi1

[GenoMax]

@Steffi: Please contact Illumina tech support who can remotely look at this run (assuming your machine is accessible over the network otherwise you will need to send them some files) to diagnose the issue.

Sounds like the machine had gone past the clustering step and must have encountered some other error. Was the cluster density number populated on the screen in MCS?

[Steffi1]

I have already contact Illumina tech support.

No, the cluster density number was not populated on the screen.

[Jessica_L]

What chemistry version and run length are you running?

Also, what version of MCS are you running? There are known issues with MCS 2.6 that can cause focusing problems on sequencing runs.

[Steffi1]

@ Jessica: I used the MiSeq v3 reagents and the length of my amplicons were between 113-112bp (without adapters, indices..). Do you think, these products/amplicons are too small to detect by the camera (i have not any experience with sequencing). I know, I can also use the MiSeq v2 chemistry, but the 2x250bp output 7,5Gb, no. of reads 15M is too little (because i need to do targeted deep sequencing).

We use MCS 2.5.0.5, do you have same problems?

[avo]

Probably worth checking a few thumbnail images to have at least an estimate if and how many clusters are present.

I would assume either massive over- or underclustering

[Jessica_L]

@ Jessica: I used the MiSeq v3 reagents and the length of my amplicons were between 113-112bp (without adapters, indices..). Do you think, these products/amplicons are too small to detect by the camera (i have not any experience with sequencing). I know, I can also use the MiSeq v2 chemistry, but the 2x250bp output 7,5Gb, no. of reads 15M is too little (because i need to do targeted deep sequencing).

We use MCS 2.5.0.5, do you have same problems?

MCS 2.5.x should be fine. Illumina had issued a notice some months back about a problem with v2.6 that was causing focusing issues, so I wanted to make sure that wasn't causing your failure. The amplicon size without any adapters or indices also sounds okay to me.

I know some users have reported issues with the 2x300 v3 chemistry, but it sounds like you're experiencing a failure at cluster generation or first base incorporation, which would be something altogether different.

Assuming the run stopped in the first cycle and you don't have thumbnail images to share, can you at least let us know what kind of libraries these are (amplicons, but how did you prep them? Kit manufacturer, etc.)? Also, how did you quantify the finished libraries and what concentration did you load on the sequencer?

Lastly, has Illumina tech support gotten back to you yet?