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Old 06-08-2015, 01:38 PM   #1
ksawatzki
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Default 2x300 MiSeq read 2 failures due to reagent?

We have been running many libraries with a core using the PE 300 kit on their MiSeqs. We've been working with them successfully for more than a year, and I've been using a set protocol for at least a couple successful runs.

Two months ago, they ran three of our libraries and read 2 (read 3, technically, we have indices) was completely unusable. Quality was well below 20 within just a few cycles. Read 1 was otherwise normal. Unfortunately for our project, our required reads are >300bp so we require both reads. I was immediately concerned that something was wrong with the adapters or index mixes, but they informed us that they saw the same problem among multiple samples. They said that it was a problem with Illumina's reagents. I have no reason to doubt them, but I didn't see anything here on a cursory search and they still haven't sequenced anything.

Does anyone know about this? What happened and when is it expected to be fixed? We're in a huge time crunch as our money source is about to cut us off and I have a ton of libraries that need sequencing. Any information and help would be appreciated.

Last edited by ksawatzki; 06-08-2015 at 02:33 PM.
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Old 06-08-2015, 04:04 PM   #2
Bukowski
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Yes this is a reagent issue. Illumina have acknowledged this. Contact Illumina. You should receive replacement reagents.
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Old 06-08-2015, 04:06 PM   #3
ksawatzki
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Great, that's very reassuring to hear. I just couldn't find anything that confirmed there was a known problem, the core tells me that they have it handled. Thanks!
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Old 06-08-2015, 06:08 PM   #4
GenoMax
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@Bukowski: Do you know that this is a widespread problem that affects all MiSeq reagents produced during some time period (do you have the lot # that were affected?). I don't remember seeing anything from Illumina Support in the last couple of months.
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Old 06-09-2015, 06:31 AM   #5
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We did a 600 cycle run about a week ago. Different failure mode. Read 1 prephasing was sky-high but phasing was low. (Read 1 0.062/0.974; Read 4 0.299/1.127) After 80 bases quality dropped and the error rate rocketed to 70%. Tech support had us do a volume test, etc. All passed. Obviously we asked that Illumina check the lot # we were using. But they said there was no issue with that lot. And maybe it was just the cassette that was bad.

So another run was started, same instrument. Pretty much the same thing happened. (Read 1 0.080/0.475 Read 4 0.349/0.667 phas/prephas %)

The libraries were customer created amplicons -- but a mixture of ~9 loci. So while there was library bias, it was minor compared to the single-plex amplicons we often run.

After the 2nd failure Illumina instrument tech support wanted a phiX 50 cycle run done on the instrument and Illumina library tech support to contact us. I asked if they thought there were library issues that could cause extremely high pre-phasing. Also the phiX and 50 cycle cassette didn't ship because none were not available that day. At which point they told us they were sending out an engineer. He arrived the next day, found some components he didn't like in the instrument and replaced them.

Meanwhile we repeated the run on our 2nd MiSeq using replacement reagents. This run looked as good as any v3 run we've ever done. So the libraries themselves were not the issue.

However, one interesting tidbit of information: tech support told us:
Quote:
In general for sequencing runs on all Illumina platforms, library composition can have an effect on the reported phasing/prephasing metrics. When the base diversity is not well balanced, it can cause issues for the software to accurately estimate the phasing/matrix information. When sequencing these types of libraries, the raw cluster density should be between 500k/mm2-1000 or 1100k/mm2 for the software to accurately generate a cluster map and extract metrics.
I'd never heard of a lower end issue -- unless you were really low, like below 200. I don't actually think this had anything to do with our issue, but I can't rule it out. I had noticed the cluster densities were low on the first 2 runs. (477 and 305 for run1 and run2). Then I noticed the person doing the runs had an error in their concentration calculations. So the 3rd run was 772.

In situations like this, my tendency is to suspect what I call "multiple systems failure". Aka "When it rains, it pours." That is, most stable systems have a robustness that tolerates 1 or 2 minor issues without substantially degrading the quality of data being collected. So when you do start seeing issues, they don't usually have a single cause, they are the result of several factors falling below optimum.

Here are the culprits I would tend to suspect for this issue:
(1) Aging MiSeq components.
(2) Cleaning/maintenance protocols for MiSeq insufficient. Possibly resulting in biofilms in some percentage of instruments.
(3) Systemic issues with v3 MiSeq reagents causing them to perform slightly below spec most of the time and substantially below spec on occasion.
(4) And the usual reporting/confirmation bias. Few people jump on a forum like SeqAnswers to say how smoothly their MiSeq runs are going.

--
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Last edited by pmiguel; 06-09-2015 at 06:34 AM.
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Old 06-10-2015, 07:33 AM   #6
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Hm, that's concerning. I'm running a 2x300 v3 run at the moment, and now, after ~500 cycles, I have a sudden drop in quality. It's not completely finished, yet, but cycles 500-580 have a quality of <10. Up until cycle 500 everything was fine with >70 % >Q30. Now it has dropped to 60 % and is still dwindling. I will contact Illumina tomorrow when the run has finished.

Oh, by the way: Illumina RTA stopped working sometime during the run. I didn't close the program, however, because everything was still running. RTA calls the bases and quality during the run, so I'm not sure how I'm still getting data?

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Old 06-10-2015, 07:57 AM   #7
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@dfhdfh
I'm not sure what the range of the problem is, but I've attached the Prinseq QC output for Read2 of one of my reagent-based failed runs. It just... immediately stops working.
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File Type: jpg Read2Fail.jpg (40.8 KB, 122 views)
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Old 06-10-2015, 08:03 AM   #8
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Quote:
Originally Posted by ksawatzki View Post
@dfhdfh
I'm not sure what the range of the problem is, but I've attached the Prinseq QC output for Read2 of one of my reagent-based failed runs. It just... immediately stops working.
Can you please clarify if there is no sequence beyond that point (N's?) or just that the Q-scores effectively become zero? Does the sequence make sense (if it is present, even if the Q-scores are all 0)?
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Old 06-10-2015, 08:25 AM   #9
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I haven't taken a detailed look at the fastq since it's a complete loss for us (even though R1 is completely normal, we require both) and also because we're not involved with the sequencers themselves. However, on cursory glance, it looks like the dominant two read types are poly-C runs and N-dominated. Even reads with some nucleotide diversity pretty much are universally 0 scores. The sequences definitely do not make sense and are obviously not biological. Just to clarify, all the R2 reads are the full 300bp long.
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Old 06-10-2015, 08:34 AM   #10
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That seems to indicate some sort of an issue with reagents/instrument. Can you ask the provider if they have run an instrument test on that MiSeq and if it passed that test?

We have been running a lot of V2 lately and there are no problems. Only V3 (600 bp) run in last couple of weeks actually performed extremely well (even though it was overloaded due to a concentration estimatation error).
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Old 06-10-2015, 08:39 AM   #11
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I know that they've been working with Illumina to work things out, and after contacting a few other cores in the area, it's definitely the reagent. They're an excellent core, and I'm confident that they ran the standard tests. In addition, they're still able to successfully run non-2x300 kits on the same machine. I was getting increasingly alarmed when I hadn't heard anything out of Illumina or on this forum about a broader (though clearly not universal) issue, but now I'm very confident that's what it is and it's being fixed.
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Old 06-10-2015, 08:44 AM   #12
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I am not doubting competency of your core. I was just trying to gather additional information to see if we (and perhaps others) need to hold off running V3 (600 bp) runs till this gets sorted out.
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Old 06-10-2015, 08:50 AM   #13
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I'd contact your Illumina rep to get their opinion. It sounds like there are regions that haven't had any trouble at all while in my area, no one I've talked to is confident or currently running the kits. I also had a chat with a midwest core and their rep suggested they hold off on new orders until everything is fully QCd, but they have been successfully running their kits with no problem. If the sample is very precious and you'll deplete it, I'd personally hold off.
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Old 06-10-2015, 08:54 AM   #14
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Quote:
Originally Posted by dfhdfh View Post
Hm, that's concerning. I'm running a 2x300 v3 run at the moment, and now, after ~500 cycles, I have a sudden drop in quality. It's not completely finished, yet, but cycles 500-580 have a quality of <10. Up until cycle 500 everything was fine with >70 % >Q30. Now it has dropped to 60 % and is still dwindling. I will contact Illumina tomorrow when the run has finished.
Actually that is pretty much what all 600 cycle runs seem to do. Oddly 500-550bp amplicons seems to PANDA merge just fine, so I tended to assume that the instrument was just losing its confidence. But since the phiX error rate also tends to sky rocket at this point, I am not sure what to think.

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Old 06-10-2015, 04:03 PM   #15
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Quote:
Originally Posted by pmiguel View Post
Actually that is pretty much what all 600 cycle runs seem to do. Oddly 500-550bp amplicons seems to PANDA merge just fine, so I tended to assume that the instrument was just losing its confidence. But since the phiX error rate also tends to sky rocket at this point, I am not sure what to think.
I have had the same experience...

The quality seems to drop quite badly. For us, it's often about half way through the read. But, our libraries on 2x300b runs are almost always low diversity so I have often thought (but not yet rigorously tested) that this was a contributing factor. The apparent error rate from (from the PhiX spike-in) seems to be high, too... but, as you mentioned, the R1 and R2 reads (i.e. per cluster) assemble to each other very well.

We have been told discretely by Illumina staff that there is an diagnosed issue with some 600c kits and that we should keep an eye on the progress, and report any problems with loss of quality in the second read.

Scott.
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Old 06-10-2015, 06:07 PM   #16
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That is enough votes now.

Like Phillip/Scott, we had assumed that this was a "feature" of the low diversity libraries we get a lot. There were no complaints so there were no immediate red flags. I would say this is going on for at least 6+ months. I am going to have to go back and dig though past flowcells tomorrow.
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Old 06-16-2015, 01:37 AM   #17
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Quote:
Originally Posted by pmiguel View Post
Actually that is pretty much what all 600 cycle runs seem to do. Oddly 500-550bp amplicons seems to PANDA merge just fine, so I tended to assume that the instrument was just losing its confidence. But since the phiX error rate also tends to sky rocket at this point, I am not sure what to think.

--
Phillip
After talking to Illumina, they said it might be the library but they cannot exclude a failure of the reagents, so they sent new reagents. I went down from 1000 K/mm˛ to 700 K/mm˛ and increased PhiX from 5 % to 12 % and am now running the same library again. This costed me 8 M reads PF (down from 25 M to 17 M). I hope that it'll still be enough for the analysis.
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Old 06-16-2015, 02:45 AM   #18
Brian Bushnell
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Quote:
Originally Posted by pmiguel View Post
Actually that is pretty much what all 600 cycle runs seem to do. Oddly 500-550bp amplicons seems to PANDA merge just fine, so I tended to assume that the instrument was just losing its confidence. But since the phiX error rate also tends to sky rocket at this point, I am not sure what to think.

--
Phillip
This won't work for the amplicon reads, but for the PhiX reads, you can plot the error rates like this:

bbmap.sh nodisk ref=phix.fa in=reads.fq mhist=mhist.txt qhist=qhist.txt ehist=ehist.txt

As long as you have at least a few percent of PhiX, it should be somewhat reflective of the error rates by position of all reads. You can use all reads for this, or just the PhiX reads; the results will be similar, since non-PhiX reads won't map to PhiX. However, it will be slightly more accurate using all reads, since that will include reads that are so low quality that Illumina's software does not recognize them as PhiX.

Last edited by Brian Bushnell; 06-16-2015 at 02:49 AM.
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Old 06-16-2015, 06:26 AM   #19
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We have been told that the only deleterious effect this observation has is reduced coverage. This appears to jive with observation that there are no general problems with sequence (they seem to overlap fine). This of course does not apply in @ksawatzki's case, since there were N's in reads in that run.

If you think you are not getting adequate coverage for 600 bp V3 runs, open a ticket with Illumina tech support and contact your local FAS.

Last edited by GenoMax; 06-16-2015 at 06:30 AM.
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Old 06-29-2015, 04:06 PM   #20
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Hello everybody,

The quality drop its a real issue, we already report to Illumina 4-5 Runs, with more or less the same metrics (last one R2=52%Q30). We got cluster density from 900 to 1400 k/mm2 and the quality its the same.
They admitted that there's a quality issue with these kits (600 cycles), but they still do not know what's the problem. They replaced already 2 cartridges, and we got the same problem than the previous (different lot nr).
This is not such a big problem for re-sequencing of genomes, since essentially the reads are still usable after more trimming, but for the 16S (V3-V4) samples it is.

Cheers
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