[martinjf]

same issue here, our last 8 kits V3 2x300 runs were reimbursed (well they sent new kits but the problem is not solved....
We rechecked all runs and the problem appeared last february at least for us.
We checked three different miseq instruments, same issue.
Illumina recognizes here too that they have an issue with the reagents, no precise lot is isolated so far, even the most recent ones fail.
Around us most providers suspended their operations with V3 2x300 for now.

hopefully they will resolve this issue soon,

have faith

[toneta2013]

We're struggling with the reagent issue in Norway as well. You've all been describing the early loss of good quality data, however we also see a quality dip in R1 around bp 6 to 20. Anyone familiar with this? I've attach a file showing a typical fastQC output on our 16S metagenom data.

[GenoMax]

We're struggling with the reagent issue in Norway as well. You've all been describing the early loss of good quality data, however we also see a quality dip in R1 around bp 6 to 20. Anyone familiar with this? I've attach a file showing a typical fastQC output on our 16S metagenom data.

If this was a one time observation it could be because of a glitch with the sequencer (e.g. small bubble in liquid path). If it is reproducible across runs, you may have a product that has low nucleotide diversity around that region seen in R1. Comments?

[toneta2013]

This happens across all runs (4 up to now), so I assume it's not a bouble. We're running the standard 16S metagenome protocol from Illumina, so I guess our diversity shouldn't be much different from anyone else's running this protocol?

[Brian Bushnell]

It sounds to me like a diversity issue, since the lowest diversity will be near the beginning. Are you using staggered primers, or spiking in anything with more diversity? There's also cluster density; even if you follow the same protocol, a higher cluster density will suffer more from a lack of diversity.

[pmiguel]

It sounds to me like a diversity issue, since the lowest diversity will be near the beginning. Are you using staggered primers, or spiking in anything with more diversity? There's also cluster density; even if you follow the same protocol, a higher cluster density will suffer more from a lack of diversity.

Brian,
Diversity should no longer be an issue with MiSeq runs. Or HiSeq runs for that matter. Modern versions of the Control Software for either instrument recognize low bias sequence and correct for it.

You just need enough phiX library (or other high diversity library) to put a few clusters per image (5-10% is fine) to keep the focus on track.

That said, and even though I've seen plenty of zero diversity MiSeq runs that look just fine, I'm suspicious at this point that the instrument might be thrown off by low-but-not-single-plex diversity. Since I've seen some poor results from sample with 4-6 amplicons. Or samples that start off single-plex but then become diverse.

But Illumina replaced the reagents for those runs. We just need to get new library pools from the customers to try again. (These were 500 cycle reagents and had a different failure mode -- good sequence for about 100 bases of each read, followed by terrible sequence quality.)

--
Phillip

[james hadfield]

At Illumina's UK UGM the team discussed the low quality at the end of long-read MiSeq kits. They acknowleged there was an issue and that they were actively working on it. However they have no plans to issue a quality notification until they understand the root cause. This issue has been rumbling on for months, at least a handful of invited users had not even heard of the problem.

Users need to be aware that 600 cycle kits are most likely not to work for long amplicons as the ends of both reads will drop in quality - badly.

The sentiment at the meeting was that issuing a quality notification to make sure customers are aware of this risks damaging confidence in these kits. Whilst many users will be unaffected (small random fragment genomes) or anyone sequencing under 500bp, the kit is clearly not delivering as expected in many long-read situations. Shareholders and investment banks may react negatively to a PQN, but without it too many users are in the dark.

Speak to your FAS and Sales team.

[toneta2013]

It sounds to me like a diversity issue, since the lowest diversity will be near the beginning. Are you using staggered primers, or spiking in anything with more diversity? There's also cluster density; even if you follow the same protocol, a higher cluster density will suffer more from a lack of diversity.

The run previously shown was run with a 4 pM 16S-library, 10 % PhiX spiked in. This resulted in a cluster density of 869. Are there anyone out there running the Illumina metagenomics protocol? Could you please provide a snapshot of a Fastqc-report from a successful run?
We are in dialog with Tech Support that are constantly sending us replacement kits and try to convince us that there do excist kits that are working... But it is frustrating to repeat runs that we assume will give crap results. And as long as Illumina do not officially acknowledge their trouble, I guess we will not be told when everything are back on track again...

[nucacidhunter]

In addition to read quality there are problems with consistency as well. Re-running the same library with the same input gives different cluster numbers. The problem definitely is worse with 16S amplicons. High diversity libraries read quality are better but not as good as they used to be or up to the specification.

[DRYTCYV]

My idea is that the missing information about the quality problem it's a little bit naive for a big company like Illumina? I'm just saying this because its impossible to change something in a procedure, in this kind of companies, and say that they don't know about this.

In our experiments looks like that even in small amplicons (metagenomic protocol), users are complaining about the low quality at the ends.

[Brian Bushnell]

Could you please provide a snapshot of a Fastqc-report from a successful run?

Here is the quality histogram for a recent 2x300bp 16s sequencing run. It's V3/V4 or V5/V6 or something like that.



The quality is very low and causes a terrible merge rate despite the fact that they are fully overlapping. "linear" is what fastQC reports; "log" is the quality based on the actual expected error rates; and "measured" is based on mapping (we have the reference genomes for this metagenome).

[Moorepark]

Hi all, have not read all the messages but get the overall stress! We have had problems with 600 cycle since approx Jan.We run primarily 16S libraries,quality on 600 used to be ok (in spec anyway, and we had such high read no.s we could afford to filter out some poor quality).Around Jan, started dropping, subtle change at first, but by May 2015 Read 4 (=read 2 we have dual indicing) was down to ~50% >Q30 and joining efficiencies were non-existant. Read 1 also poor. We have been engaging with Illumina on this since March and it is definitely a reagent issue, Illumina admitted as much to us and we've abandoned 600 cycle since then.We tried running low sample number, and included amplicons from good runs in the mix, but read numbers are so low data is unreliable and not consistent with previous good runs.Also tried increasing phi X etc but with increased phix and decreased sample, read numbers were too low so abandoned 600 cycle pretty much. we're running 500 cycle V2 since then. Apparently those kits also effected but not as badly, most have been fine for us. We ran one library on out of date kit and got average Q30 at 82%, same library two days later on new kit- quality at 69%- so there is an effect, but we still got enough data.most 500 cycle higher than that anyway. My big thing I want to say is I was just at a Users meeting in the UK. We asked why Illumina had not yet made a public announcement on this and were told "There will be no public announcement until we have 100% identified the problem and removed it". I.e. they're still making money selling the kits and it's cheaper to replace the ones from people who complain than withdraw everything. Problem for them is the issues are not even consistent, some people also seeing awful index reads but ok everything else, sometimes we get really high CD when we know we loaded similar amounts to previous....a lot of strange things going on...bottom line, you run a bad 600 cycle, go straight to them and say you want replacements, and switch to 500 if you can. A lot of people at meeting were not running 600 cycle routinely, so when they got a bad run, they blamed library and didn't pursue it, they were shocked to hear this is going on almost a year! We also need to put pressure on for public announcement because for those who have to use 600 cycle, an announcement would get funding agencies off their backs.

[toneta2013]

Hi Moorepark, it's good to hear that the 16S runs perform better with the V2 500 kit. Tech support has offered us to replace V3 600 kits with the V2 500 and also provide us with more Indexing kits. What fragment length do you run on the 500 kit? Would it be possible to get information regarding which protocol you're using? We are working on an EU financed project with deliverables by the end of the year, so we're pretty desperate...

[Moorepark]

hi,we follow the Illumina 16S metagenomic method-so fragment is about 430bp,we thought it would be too short for overlap with 2 x 250bp but actually we're getting good joining efficiencies. It's not ideal, but more trustworthy than the garbage 600 cycle. We run less samples (usually 50 instead of 100) to compensate for lower output and we've included amplicons from previous runs to make sure clustering as before etc....We have one client though with longer amplicon (>500bp) and we can't help at all....it's embarrassing trying to explain when Illumina won't make an announcement. Hope that helps!

[d00b]

V2 kit is better than V3

Qscore dropping

http://postimg.org/image/zfwnk5r6n/