Question About Illumina Paired end Sequencing

[Dr. Hybe]

Quick question? Would it be possible to reposition the flowcell between read 1and 2 on the GA2x? i.e. take the flowcell/prism off and clean it in between reads? Also would it be possible to load the flowcell and do a manual fluidics inspection b/w R1 and R2? What about recalibrating b/w R1 and R2?

Thanks in advance for your response,

Dr. H

[kmcarr]

Removing a flow cell between read 1 and 2, no you absolutely can not do this IF you want to maintain the pairing between the reads which I presume is a requirement. The location of clusters relative to the 0,0 point of the flow cell coordinate system is determined during the first 5 cycles of read 1 and is thus set for the remainder of the run. It's not within the realm of human ability to reposition the flow cell to within the tolerances required (at least speaking for me, I shouldn't assume for anyone else I suppose )

[GW_OK]

I think you can do it on a Hiseq but on a GA, as kmcarr said, don't do it if you want to keep the pairings.

[kmcarr]

I think you can do it on a Hiseq ...

Yesterday I would have believed there was no way you could do this on the HiSeq either, under the same theory as the GA. Our FSE just happened to be here working on our GA so I put the question to him. Yes, you can remove and replace the flow cell on a HiSeq at any time during the run. Naturally Illumina does not recommend this and should only be done in extreme circumstances. The HiSeq flow cells have registration marks on them which the software can use to provide a precise relative positioning. If a flow cell is removed mid run, when you remount the flow cell the software locates the registration marks and resets the 0,0 position according to those.

Learn something new everyday. Thanks SeqAnswers and GW_OK.

[Dr. Hybe]

Thanks for the replies - Also I learned to day from FSE that it is possible on the GA to reload the calibration specifications in case you need to Load flowcell to check fluidics or shut down the software for some reason. Again not recommended, but possible if you have to. It will be interesting to see if they put markers on newer versions of the GA flowcells...

[Santosh]

Focus Issue

Hey all, here we have a new problem. We are running a 101 bp Paired End run. The Read 1 was done and the Read 2 had also started. After reading through 117 bases, there was a power shut down and everything including the machine, stopped working. It happened while the 117th Base Scanning was half way through. The run was anyway resumed after 10 minutes and the scan mix was pumped. The scanning started from the 1st tile of the 117th base. But then, only the right half of the tile was showing clusters and the exact left half was completely dark. Could that be some kind of Focus Issue?

[Santosh]

Here I have another question. Just imagine, the Paired End run is going on and the machine is somewhere in Read 2 and ran out of focus. If u can stop the run and then go back to the calibration tab before the Read 1 FBR, and then resume the run from Read 2 after the Calibration, will it impact the coordinates on the flow cell? Just out of curiosity, will it have the the same calibration metrics as earlier? Because essentially here, we dont have to move the flow cell from its stage. Its just the Recipe we have to play with.

[GW_OK]

How did you resume your run? Did you see if the camera did any alignments to the flowcell? Also, a total power outage is quite likely to cause vacuum loss on the flowcell, I'd check that out pronto.

PS, the same thing happened to us but we were out for 30 minutes. Total loss of vacuum.

[HESmith]

We've been able to recover the run after an "intential" power outage (b/c of port comm. issues, we had to restart the computer and HiSeq). We resumed the run from the start screen, and went through the normal checks of vacuum (which had been lost) and flow. The data (paired-end 76bp) were fine.

[Santosh]

Hi,
Thanx fr the reply.
But referring to what GW_OK said about the vacuum loss, is it applicable to GA2X? I thought vacuum operates only in Hiseq. And also, after resuming the run, we straight away resumed the run by pumping 75 uL of SMX. Even the camera did not do any kind of alignment on the flow cell. It straight away started imaging from the first tile of the cycle where it stopped.

[GW_OK]

Oh, yeah, that vacuum loss is only for the Hiseq. It might be some sort of focus issue with the GA2, or the stage might have moved minutely out of position, I don't know. I think there is a way to start the scan over completely for a step but I can't remember, that way it wouldn't start right at the tile it left off.
Anyway, how did the rest of the run look?