[DRYTCYV]

Illumina have advised us not to run 2x300 on these cartridges and to only use them for 2x250 runs. If that doesn't generate enough coverage for our application they will then send replacement reagents.

Has anyone else recieved this advice?

Yes, same thing here

[dfhdfh]

Yeah, it's a shame.

[Bardj]

Same Issues in Buffalo

We've been running into major issues joining 16s reads for the v1-v3 region. Quality drops like a rock as most of you are also talking about. 2x300 PE MiSeq. They applied the "optical fix" but that did not help.

They have been reimbursing all of our MiSeq kits, and we were told the same advice - sequencing until you get enough data and they will reimburse. Unfortunately we need the full length 2x300 so that will not work for us.

[Bukowski]

Illumina have advised us not to run 2x300 on these cartridges and to only use them for 2x250 runs. If that doesn't generate enough coverage for our application they will then send replacement reagents.

Has anyone else recieved this advice?

[DRYTCYV]

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

[GenoMax]

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.

[Brian Bushnell]

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.

[dfhdfh]

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/mm² to 700 K/mm² 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.

[GenoMax]

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.

[ScottC]

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.

[pmiguel]

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.

--
Phillip

[ksawatzki]

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.

[GenoMax]

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.

[ksawatzki]

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.

[GenoMax]

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).