Software Malfunction? (2.5.3)

[Anthony.287]

Hello again!
New question – During the most recent sequencing run, the GS software (2.5.3) stopped responding after about two hours. That was two days ago. The run finished successfully, and the data pushed to our data rig, but the software is still frozen. I’ve also had a problem where the software doesn’t display the progress bar at the top and doesn’t display the raw wells, keypass wells, or control wells numbers until the run is complete. Has anyone else experienced this?
Thank you!!

[sklages]

You should contact Roche support for that. Why 2.5.3, not 2.6?

[MissDNA]

We are reprocessing several of our runs using v 2.6 and we got mora data than when analyzed with v 2.5.3.

[sklages]

That's good, isn't it? ;-)

Probably due to better CAFIE correction in 2.6.

[MissDNA]

Very! That is why I decided to share the news, so someone might consider reprocessing as well.

It has added several Mbp to our data and assembling has improved as well.

[Anthony.287]

Thanks for the info! I was going to ask if there were any advantages to upgrading to 2.6, and it seems that there are. 1 question - we've done a lot of 16S Amplicon runs w/ 2.5.3; we won't be able to analyze those runs with 2.6, will we?

[MissDNA]

I would think you can since Amplicon runs are still supported using XLR70 chemistry. We keep using XLR70 kits with our upgraded machine and sw. The data I said was improved, was obtained using XLR70.

Do you analyze your amplicons 16S with SG or amplicon pipeline?

[pmiguel]

I just wanted to add that we frequently see the our instrument control software enter a non-responsive state for our GS-FLX. In one case this occurred early in a run and the entire run was lost because of it. GSSupport said the fix was to completely power the instrument down for at least 15 minutes, then bring it back up. After this it should not enter the non-responsive state.

We found this to be the case -- during the run. However when starting a wash or starting a run we frequently see a possibly related issue: the instrument refuses to register that the module that holds the sipper tubing has been lowered again after raising it. This also seems to be accompanied by the software more-or-less freezing. (Some things work, but most buttons are non-responsive.)

Again a complete shut-down for >15 minutes appears to cure this behavior for some time.

Finally, I should mention, for those of you that have never shut the instrument completely down (including powering it down using the rocker switch on the right side of the instrument) you might want to have tech support walk you through it. The confusing part is that after doing the operating system/computer shutdown, you can power down the rest of the instrument using the rocker switch on the right side of the instrument.

However, when you power the instrument back up with the rocker switch, the computer does not come back up automatically. Instead you have to remove a grill on the front of the instrument covering the front panel to the server. Then you need to depress a small button you will find there. I use a 1000 ml pippette tip to do it. Then the server makes a strange whistling/roaring sound as it powers back up.

Then, after waiting a while for the login screen to come up, you login. You can then launch the control software. But if you actually wan to start a run, you will need to wait for the CCD to cool back down. This takes >10 minutes in my experience.

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Phillip

[Anthony.287]

Great info, everybody! Glad to see that I'm not the only one experiencing this issue with the software..
As for the question, lately I've been analyzing every run with both the shotgun and the amplicon pipelines, and giving the customer both sets of data. I'm going to analyze it both ways strictly out of curiosity, and the customers don't seem to mind!

[MissDNA]

One time one customer asked for shotgun analysis in a 16S experiment because there was more reads. When he compared both pipelines we opted to use amplicon pipeline.

Anthony, what have you been observing? Do you think analysis done with SG pipeline is trustworthy?

[Anthony.287]

Phillip,
The last time I spoke with a Roche FAS, about the progress bar and the raw, keypass, and control wells number, I was told that many users reboot before every run or once a week to avoid these software problems. Wish we would get this information before we lose runs!
Most of the time I see more reads with the shotgun pipeline. I wish I could see more details about the failed sequences; especially the short quality. It would be very useful for troubleshooting, especially considering we have no FAS.
As far as the customers, I think I only have one or two customers with real bioinformatics people that would be able to tell the difference. I have one customer doing 16S with the Broad primers, and that protocol calls for the shotgun pipeline. I’m really not the best one to ask about this, but I don’t see why the shotgun pipeline would be less than trustworthy. The great thing about both of them is that you can tweak the settings, although I have not had the time to try that.

[MissDNA]

Honestly my knowledge in bioinformatics is pretty much null, however we have a good team of bioinformaticians, and I heard from one of them that SG pipeline was bad for 16S analysis. I don´t recall the details but it had to do with filters.

[maven]

Whether or not shotgun or amplicon is more appropriate for 16S has a lot to do with how diverse your 16S sample happens to be. Maybe yours isn't that diverse, and so isn't that shotgun-like.

[pmiguel]

Phillip,
The last time I spoke with a Roche FAS, about the progress bar and the raw, keypass, and control wells number, I was told that many users reboot before every run or once a week to avoid these software problems. Wish we would get this information before we lose runs!

From what I was led to understand, a reboot would not necessarily be sufficient to avoid this issue. It would require a >15 minute power down. Of course I was told that it should not re-occur after doing this once. So we may have a separate issue.

--
Phillip

[Anthony.287]

I will now add a >15 minute power down to the protocol. Thank you!!
Seriously, thank you. You guys are my go-to tech support!!