I made a cursory attempt to use RTA to re-process a run from the image files (.tif). I'll go into the whys and wherefores of doing such a thing below. I just wanted to note that in my hands nothing productive came out of it.
Just in case my description might be of use to future explorers of this brave new realm:
It was easy to find the directory where RTA resides. Double-clicked on it and the GUI launches. I point it a the image directory I want process. I hit start. RTA tells me that run has already been processed and declines to run again. I tried isolating the images away from the previous processing directory, but I am kind of at sea here. No idea what files are needed. So I just tinker around with some of the config files until RTA agrees that there is a run to process. One major problem: it mysteriously displays 32 panels/lane instead of the v3 48. Since the run will be repeated once the reagents arrive anyway, I decide to just start the processing anyway. I let it run for ~16 hours. It does seem to be doing something, but nothing useful. So I terminate the processing.
I have some ideas as to how one might proceed from here. Illumina tech support is not eager to guide me. They offered the older version of OLB, that still did image analysis. Rick gave that a shot and it did not look like it was going to be productive either. But, I still think RTA might be cajoled into actually processing image data again. Maybe one could create a faux run to build the right file/config file structure, then link the old image data there and point RTA at it.
Okay, why would anyone want to do this? Up to some point it was possible to do image analysis offline. This provided one avenue to avoid Illumina's Achilles's heel: lack of what I'll refer to as "variety" in the first 4 cycles causes cluster calling to work poorly or fail altogether.
Might as well upgrade the above statement to a full denunciation: Illumina, you get many things right, but you get a big "FAIL" on this issue. The work-around is to make sure there sufficient "variety" in cycles 1-4 of your library. But, c'mon, that is a dim-witted way to build a system!
So, a secondary work-around was developed for the Genome Analyzer where you re-do image analysis on a re-ordered set of images. That is, you have the software do cluster calling starting at a later cycle of your run -- past where the low "variety" sequence is. Then restore the actual order of the cycles later on.
I came to the Illumina party only recently, so I never partook of this practice (called "GOAT-fooling, etc.) Plus, initially it was stated that it would not work on HiSeqs (or HiScanSQs, by extension) because save the images was not possible.
But it is possible to save the image files. Not necessarily wise (especially if you are storing them locally), but possible. So, I thought it might be easy just to fire up RTA and re-process a run, should the need occur. And, if this is possible, then fooling RTA would likely be possible as well.
Anyway, as it turns out, I did not have a cluster-calling issue that drove me to give it a try. I had, well what would you call it? Basically some issue caused high pre-phasing for 2 lanes of a run. One of these lanes was our phiX control lane. I am not sure what the benefits of specifying a control lane for a run is, but the down-side is that if anything goes wrong with that one lane, your run is hosed. So, our run was hosed, even though only a single data lane had the pre-phasing issue. Quality values take a dive around cycle 30 despite all other metrics looking good for lanes 1-6.
So, this is not a big deal, if you have your "intensity files" saved. You just fire up your OLB (Off-Line Basecaller) and specify no control lane. Not sure why that "save intensity file" box did not get checked for this run. I mean we were saving the image files, why not the intensity files as well? Anyway, I thought it should be possible to re-generate the intensity files through RTA. And, as I say above, I still think it is possible. But not trivial enough for me to pursue it further at this time...
(BTW, I don't want to appear to be hammering Illumina too hard here. Their tech support actually works well and they are replacing all the run reagents. Given the alternative, that counts for a lot.)
--
Phillip
Just in case my description might be of use to future explorers of this brave new realm:
It was easy to find the directory where RTA resides. Double-clicked on it and the GUI launches. I point it a the image directory I want process. I hit start. RTA tells me that run has already been processed and declines to run again. I tried isolating the images away from the previous processing directory, but I am kind of at sea here. No idea what files are needed. So I just tinker around with some of the config files until RTA agrees that there is a run to process. One major problem: it mysteriously displays 32 panels/lane instead of the v3 48. Since the run will be repeated once the reagents arrive anyway, I decide to just start the processing anyway. I let it run for ~16 hours. It does seem to be doing something, but nothing useful. So I terminate the processing.
I have some ideas as to how one might proceed from here. Illumina tech support is not eager to guide me. They offered the older version of OLB, that still did image analysis. Rick gave that a shot and it did not look like it was going to be productive either. But, I still think RTA might be cajoled into actually processing image data again. Maybe one could create a faux run to build the right file/config file structure, then link the old image data there and point RTA at it.
Okay, why would anyone want to do this? Up to some point it was possible to do image analysis offline. This provided one avenue to avoid Illumina's Achilles's heel: lack of what I'll refer to as "variety" in the first 4 cycles causes cluster calling to work poorly or fail altogether.
Might as well upgrade the above statement to a full denunciation: Illumina, you get many things right, but you get a big "FAIL" on this issue. The work-around is to make sure there sufficient "variety" in cycles 1-4 of your library. But, c'mon, that is a dim-witted way to build a system!
So, a secondary work-around was developed for the Genome Analyzer where you re-do image analysis on a re-ordered set of images. That is, you have the software do cluster calling starting at a later cycle of your run -- past where the low "variety" sequence is. Then restore the actual order of the cycles later on.
I came to the Illumina party only recently, so I never partook of this practice (called "GOAT-fooling, etc.) Plus, initially it was stated that it would not work on HiSeqs (or HiScanSQs, by extension) because save the images was not possible.
But it is possible to save the image files. Not necessarily wise (especially if you are storing them locally), but possible. So, I thought it might be easy just to fire up RTA and re-process a run, should the need occur. And, if this is possible, then fooling RTA would likely be possible as well.
Anyway, as it turns out, I did not have a cluster-calling issue that drove me to give it a try. I had, well what would you call it? Basically some issue caused high pre-phasing for 2 lanes of a run. One of these lanes was our phiX control lane. I am not sure what the benefits of specifying a control lane for a run is, but the down-side is that if anything goes wrong with that one lane, your run is hosed. So, our run was hosed, even though only a single data lane had the pre-phasing issue. Quality values take a dive around cycle 30 despite all other metrics looking good for lanes 1-6.
So, this is not a big deal, if you have your "intensity files" saved. You just fire up your OLB (Off-Line Basecaller) and specify no control lane. Not sure why that "save intensity file" box did not get checked for this run. I mean we were saving the image files, why not the intensity files as well? Anyway, I thought it should be possible to re-generate the intensity files through RTA. And, as I say above, I still think it is possible. But not trivial enough for me to pursue it further at this time...
(BTW, I don't want to appear to be hammering Illumina too hard here. Their tech support actually works well and they are replacing all the run reagents. Given the alternative, that counts for a lot.)
--
Phillip
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