[aleferna]

I have the same problem here

[yaximik]

This may happen if in previous (erroneous) run Z motor hit the bottom of the moving range and cannot recover. Quitting an restarting the software will reinitialize everything including homing Z motor.

[matth431]

I just had a similar "Object reference" failure about 185bp into read 1. In our case, cluster density was very low (Nextera XT normalised libraries pooled and diluted as per instructions but according to user, library concentration prior to going into normalisation step sounds low - 1-5ng/ul compared to 20-30ng/ul with libraries I've prepared myself).

Density was only 133K/mm2 which is way lower than our worst run to date (min 700k/mm2). We typically run 900-1300k/mm2.

Thanks for the tip on getting the MiSeq to analyse the data from the failed run, though - user wants to have a look at it although without the index reads I suspect it will be fairly meaningless.

[yaximik]

Did you try to restart the run using the same cell and reagent set? It worked several times, I just used shorter run times and even got paired read completed. The reads have a lot of Ns and low quality regions, but with aggressive cleaning substantial set of useful data can be recovered. My feeling is sometimes BioAnalyzer traces are misleading in regard to average library size as contamination with shorter fragments can be significant yet not clearly visible. That results in a large portion of clusters being shorter then anticipated and vertical focus unexpectedly fails hitting the floor. That is usually signified by steep declines in the signal intensities on the graphs.

[matth431]

Did you try to restart the run using the same cell and reagent set? It worked several times, I just used shorter run times and even got paired read completed. The reads have a lot of Ns and low quality regions, but with aggressive cleaning substantial set of useful data can be recovered. My feeling is sometimes BioAnalyzer traces are misleading in regard to average library size as contamination with shorter fragments can be significant yet not clearly visible. That results in a large portion of clusters being shorter then anticipated and vertical focus unexpectedly fails hitting the floor. That is usually signified by steep declines in the signal intensities on the graphs.

I wasn't aware it was possible to restart a failed MiSeq run? Surely it rejects the cartridge and flowcell based on the RFID tags?

[yaximik]

Here is my earlier post how to do this.

http://seqanswers.com/forums/showthread.php?t=25371

If the run has aborted during read 1, then reagents are OK. However, if run terminated normally, either SE or PE, then reagents cannot be reused as MiSeq spits back something (backflush?) and ruins remaining reagents. What a waste!