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Old 11-30-2012, 07:06 AM   #1
yaximik
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Default Rescuing stopped MiSeq run

In addition to my earlier post on recovering data from a stopped MiSeq run, I want to share a method of rescuing the entire run. Basically, if MiSeq quits on you during read 1 phase (SE or PE run), here is a chance to get around the problem and salvage the kit.

1. Find the last cycle number, analyze intensity and quality plots and decide at what number you are comfortable to stop the run
2. Get RFID bypass code from ILMN. It is good for a week.
3. Start pos-trun wash as suggested by the software. If it is not suggested, quit the control software and restart it, then perform wash in maintenance. It is a good idea to restart software anyway as hardware needs to be initialized and everything homed after run was aborted.
4. Pull out the reagent cartridge and the PR2 bottle. Pluck out the RFID chips from both.
5. Aspirate contents of wells 5 (AMS1), 9 (LMX1), 17 (your library), wash with water, refill with water.
6. Add more read 1 primer (custom or standard) diluted as recommended in the manual, refill water wells.
7. If post-run wash is complete, pull out the flow cell. Carefully remove plastic cover, pluck out the RFID chip, blot excess water and snap in the cover back.
8. Edit the sample sheet (using either Excel or text editor) of the failed run - modify the reagent kit barcode (I just increment it by 1), number of cycles (SE or PE) as decided earlier and save under the name of the modified kit barcode.
9. Start new sequencing run. If it complains about unfinished analysis of the previous run, terminate analysis - use the procedure I described to get data out.
10. Enter new barcodes for the flow cell, the reagent kit (matching that entered in the modified sample sheet) and PR2 using the RFID bypass code. I just slightly modify the originals, like adding 1 somewhere. Make sure you specify the correct sample sheet.
11. Hit the run.

Basically the idea was to mock the cluster generation phase and start run 1 anew. In all cases I tried so far, the procedure worked both for SE and PE runs. The only problem is that, while most reagents remain in sufficient quantities, LPM (well 7) and RMF (well 11) may dwindle below sufficient. Any ideas what those could be? I guess the only way, until their composition becomes known, is to save excess of those from used reagent kits. Keep in mind though, that v1 and v2 may not be compatible as different abbreviations are used for some reagents.
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Old 04-25-2013, 02:08 AM   #2
JPC
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Hi Yaximik, thanks for posting this protocol, could you explain a little about point 1 please?
"decide at what number you are comfortable to stop the run"...I would only go to this protocol if the run had already stopped so how can stopping the run be the first step?
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Old 04-25-2013, 04:17 AM   #3
GenoMax
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Yaximik: I appreciate your efforts to document ways of recovering stopped MiSeq runs but it is not clear if this method is approved by Illumina. If it is not then you should add a disclaimer to the post that clarifies this.
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Old 04-25-2013, 07:19 AM   #4
yaximik
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Quote:
Originally Posted by JPC View Post
Hi Yaximik, thanks for posting this protocol, could you explain a little about point 1 please?
"decide at what number you are comfortable to stop the run"...I would only go to this protocol if the run had already stopped so how can stopping the run be the first step?
Everyone who find this useful is welcome. This line only applies if you miscalculated the usable number of cycles and the MiSeq quits on you because its optic hits the floor failing to focus. This happened to me several times prompting to invent this workaround salvaging expensive kit. I do not know reason for this and ILMN tech support was not helpful either. Signal intensity curves were of bell type and followed by a sharp decline in signal intensity right before software crashed. When salvaging the the failed run, from these curves one can estimate where to stop the run by specifying a lesser number of cycles than caused the crash. Like I said, I still have no idea what caused the problem as libraries were of sufficient length according to BioAnalyzer.
This does not apply if the run crashed in very beginning, for example it happens when the above error caused the crash. Resuming the run without restarting the software, or better MiSeq, may result in failure to home Z motor, so run crashes without making a single read. The clusters are still OK, so restarting MiSeq allows to salvage the run as described.

As to GenoMax comment, of course it s not endorsed by ILMN. If you lucky and get a good representative who submit to your cry, they will send a replacement kit. I hit one quite stinky guy, let us not finger, which was adamant - that is your problem. So I said screw you stinker and salvaged the run.
Again - you can salvage either SE or PE only if the run has crashed BEFORE flip over is done.
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Old 03-26-2014, 10:53 AM   #5
DJ Flowcell
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Default Edit Protocols in C: Drive

Yaximik's outline to bypassing the system RFID checks is a good way to do it. An alternative way to restarting the run is to edit the protocols found locally in the system; C: Drive>Illumina>MCS>Recipe>V1,V2,V3>Default,Amplicon>Protocol

I suggest copying the file you'd like to edit for later restoration. You can then remove the "OnBoardClustering" region in the script so it jumps straight to "FirstReadPreparation". However, doing it this way will require you to top off 3 reagents: incorporation buffer, scan mix, and cleavage mix
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Old 12-20-2015, 07:36 AM   #6
MasterSeq
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Unhappy What does "topping off" mean?

Ok, I know what it means, but how does one do it? Collect leftover reagent from used reagent cartridges? "Borrow" from a new cartridge?

My run stopped after clustering. Message was "No usable signal found in the images; it is possible that clustering has failed. (Illumina.Instrument.Bolt.Logic.AutoFocus.DarkSampleException)"

Thanks for your help!
Not-so-MasterSeq
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Old 12-21-2015, 08:22 AM   #7
Jessica_L
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yaximik-
I'm not sure this is what you meant, but here's what I know:
LPM is the Linearization PreMix. It's not the actual Linearization Mix used to linearize clusters after bridge amplification at either the initial cluster gen step or the paired-end turnaround (resynthesis) so I'm guessing it's a buffer, similar to the linearization mixes, just without the enzymes. You may be able to look at the safety data sheet (SDS) to get a better idea of what the chemicals actually are in that.

RMF is the resynthesis mix, probably has a polymerase enzyme and dNTPs, etc. to synthesize complementary strands of DNA as part of the PE turn around. Again, you might be able to get some information from an SDS, but probably not exact concentrations. I'm not seeing anything online about composition or components. I have a feeling that the RMF is going to be a tricky one to deal with-- buffers are usually stable and okay to re-freeze but I've noticed that Illumina frequently recommends not re-freezing solutions that contain enzymes (i.e. IMX). Maybe that has more to do with the fluorophores than the enzymes, though.


MasterSeq-- I'd advise against borrowing reagents from a new cartridge as then the new cartridge will be short on its reagents. Plus the new cartridge would be thawed and you'd have to use it in a week (which depending on how busy your lab is, may or may not matter). It's conceivable that you could save leftover reagents, but again, solutions with enzymes may be less than stable.

I'd say in the situation of your stopped run, it sounds like clustering failed. If you're positive that your libraries are good and you spiked in some PhiX, and something should have worked, you'd be better off contacting illumina tech support-- my experience is that the issue you're seeing isn't solvable via run rescue protocols.
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Old 01-20-2016, 03:25 AM   #8
JBKri
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Quote:
Originally Posted by yaximik View Post
In addition to my earlier post on recovering data from a stopped MiSeq run, I want to share a method of rescuing the entire run. Basically, if MiSeq quits on you during read 1 phase (SE or PE run), here is a chance to get around the problem and salvage the kit.

1. Find the last cycle number, analyze intensity and quality plots and decide at what number you are comfortable to stop the run
2. Get RFID bypass code from ILMN. It is good for a week.
3. Start pos-trun wash as suggested by the software. If it is not suggested, quit the control software and restart it, then perform wash in maintenance. It is a good idea to restart software anyway as hardware needs to be initialized and everything homed after run was aborted.
4. Pull out the reagent cartridge and the PR2 bottle. Pluck out the RFID chips from both.
5. Aspirate contents of wells 5 (AMS1), 9 (LMX1), 17 (your library), wash with water, refill with water.
6. Add more read 1 primer (custom or standard) diluted as recommended in the manual, refill water wells.
7. If post-run wash is complete, pull out the flow cell. Carefully remove plastic cover, pluck out the RFID chip, blot excess water and snap in the cover back.
8. Edit the sample sheet (using either Excel or text editor) of the failed run - modify the reagent kit barcode (I just increment it by 1), number of cycles (SE or PE) as decided earlier and save under the name of the modified kit barcode.
9. Start new sequencing run. If it complains about unfinished analysis of the previous run, terminate analysis - use the procedure I described to get data out.
10. Enter new barcodes for the flow cell, the reagent kit (matching that entered in the modified sample sheet) and PR2 using the RFID bypass code. I just slightly modify the originals, like adding 1 somewhere. Make sure you specify the correct sample sheet.
11. Hit the run.

Basically the idea was to mock the cluster generation phase and start run 1 anew. In all cases I tried so far, the procedure worked both for SE and PE runs. The only problem is that, while most reagents remain in sufficient quantities, LPM (well 7) and RMF (well 11) may dwindle below sufficient. Any ideas what those could be? I guess the only way, until their composition becomes known, is to save excess of those from used reagent kits. Keep in mind though, that v1 and v2 may not be compatible as different abbreviations are used for some reagents.
Just want to add my modification to this recipe; there is no need to remove the RFID chips (they stick quite well) - just cover them with a piece of aluminum foil - that is enough to block the radio signals.
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Old 09-20-2018, 08:00 AM   #9
chelie
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Sorry for double-posting ... see below.

Last edited by chelie; 09-21-2018 at 02:05 AM. Reason: double posting
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Old 09-20-2018, 08:22 AM   #10
chelie
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Although this thread has lain dormant for quite some time, I wanted to ask whether anyone is willing to share his/her experiences with restarting MiSeq runs?

The above comments are very helpful in this respect, but I am wondering whether the introduction of the V3 chemistry might have changed anything to the recommended?

Also, I would like to know if manipulating the sample sheet might help in this respect. For example, is it possible to reduce read 1 to five cycles only (or to 0), skip both indexing reads, and leave the reverse read at its full 76 or 301 cycles?

Cheers
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