[ksawatzki]

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.

This is a huge problem for us. We lost many thousands of dollars trying to
"fix" this problem on the library prep end, and we had tens of thousands of dollars allotted to sequencing on a government contract. Now the contract is over and we weren't able to get the sequencing done in time because of Illumina. We found a facility that still had semi-working kits, but we need Illumina to either officially announce their kit problem so our contract admin has a paper trail or pay for all the parts and labor independently.

Does anyone have any idea on who I can contact at Illumina? Our local rep just brushes us off, but this is not something we're going to just allow.

[Bukowski]

Illumina have already stated they will not release a PQN for this issue, so don't expect that to change...

[ksawatzki]

Yeah, I'm hoping that they'll be willing to help us since our paper trail involves a very early recognition of the problem, which we immediately brought to their attention. Our runs have definitely been a worst-case-scenario for them - not just quality drop off after 100-200bp, but a total loss of quality within 50bp. The exact same library was run at a different facility with older versions and worked relatively well, with the more normal drop off after a >150bp, pointing to the kit as the culprit.

My main question is if anyone has a lead on a good person at Illumina to contact. I'm pretty fired up about this whole incident, I'm not even sure how ethical it is for them to just pretend there's not a problem with the public while privately telling us the kit's not working.

[d00b]

Yeah, I'm hoping that they'll be willing to help us since our paper trail involves a very early recognition of the problem, which we immediately brought to their attention. Our runs have definitely been a worst-case-scenario for them - not just quality drop off after 100-200bp, but a total loss of quality within 50bp. The exact same library was run at a different facility with older versions and worked relatively well, with the more normal drop off after a >150bp, pointing to the kit as the culprit.

My main question is if anyone has a lead on a good person at Illumina to contact. I'm pretty fired up about this whole incident, I'm not even sure how ethical it is for them to just pretend there's not a problem with the public while privately telling us the kit's not working.

Dear Ksawatzki,

Many peoples are complaining V3 kit issue.
Many seq core facilities are refusing V3.

Read these articles from everywhere.


Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform.--> http://www.ncbi.nlm.nih.gov/pubmed/25586220

A novel conceptual approach to read-filtering in high-throughput amplicon sequencing studies --> http://nar.oxfordjournals.org/conten...v1113.abstract

http://www.ncbi.nlm.nih.gov/pubmed/23793624

http://blog.mothur.org/2014/09/11/Why-such-a-large-distance-matrix%3F/

http://www.mothur.org/forum/viewtopic.php?t=3685

http://seqanswers.com/forums/showthread.php?t=65064

http://seqanswers.com/forums/showthread.php?t=40879

http://omicsomics.blogspot.com/2015/12/loose-2015-threads-1-miseq-2x300-issues.html

https://www.researchgate.net/post/why_the_Q30_of_i7282_is_much_lower_than_i5923

[Moorepark]

CEO's and presidents name are both on the Illumina website- maybe you should start at the top! If you succeed in getting any satisfaction let us know.....

[Markiyan]

Use asymetric run mode with 600 cycle kits.

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.

Actually the one should count not the number of the raw PF reads, but the number of good quality reads left after all preprocessing steps.
Sometimes the 25M PF with 0.1% passed all processing steps is way worse, than 8M PF with 15% of reads passing preprocessing.

Also it can be quite helpful to do asymmetric 600 cycle runs - add some (10-15% of cycles from R2 to R1): instead of R1:300,I:6,R2:300,

run it in the R1:330,I:6,R2:270 mode, since last 30 - 50 cycles of the R2 are just wasting reagents better spent on the R1 read.

[dannyhi321]

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.

Has anyone heard of any developments re this issue?

[dannyhi321]

This is a huge problem for us. We lost many thousands of dollars trying to
"fix" this problem on the library prep end, and we had tens of thousands of dollars allotted to sequencing on a government contract. Now the contract is over and we weren't able to get the sequencing done in time because of Illumina. We found a facility that still had semi-working kits, but we need Illumina to either officially announce their kit problem so our contract admin has a paper trail or pay for all the parts and labor independently.

Does anyone have any idea on who I can contact at Illumina? Our local rep just brushes us off, but this is not something we're going to just allow.

Is anyone currently using these v3 600 kits? Anymore feedback from Illumina?

[GenoMax]

We are using 2x300 kits. There is no feedback from Illumina. As detailed in this thread, if you feel that you did not get enough data, you can open a ticket with tech support and they may provide a free kit (this may be dependent on having a current maintenance contract).

[dannyhi321]

We are using 2x300 kits. There is no feedback from Illumina. As detailed in this thread, if you feel that you did not get enough data, you can open a ticket with tech support and they may provide a free kit (this may be dependent on having a current maintenance contract).

There IS feedback from illumina:

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.

I assume you have not had any issue with the kits ?

Do you feel this is an acceptable response to the ongoing issue?

[GenoMax]

@dannyhi321: The response you copied above was posted by @James Hadfield earlier (post #37) in this thread. I thought you were asking if there has been any feedback recently and the answer for that is no, AFAIK. Only thing we ended up doing was downgrading MCS to v.2.5.x based on recommendation from Illumina (there is a PQN for that).

We sequence all kinds of libraries (many are low diversity). Some work better than others but we have not had any outright failures. Most of the times the drops in Q-scores have been because of shorter inserts (than expected) otherwise for others we have been able to routinely get Q30 > 75%. We do spike-in 10% or more phiX based on the sample type for low diversity samples.

[dannyhi321]

@dannyhi321: The response you copied above was posted by @James Hadfield earlier (post #37) in this thread. I thought you were asking if there has been any feedback recently and the answer for that is no, AFAIK. Only thing we ended up doing was downgrading MCS to v.2.5.x based on recommendation from Illumina (there is a PQN for that).

We sequence all kinds of libraries (many are low diversity). Some work better than others but we have not had any outright failures. Most of the times the drops in Q-scores have been because of shorter inserts (than expected) otherwise for others we have been able to routinely get Q30 > 75%. We do spike-in 10% or more phiX based on the sample type for low diversity samples.

Do you think no feedback re the reagent issue is acceptable?

[GenoMax]

Do you think no feedback re the reagent issue is acceptable?

This has not been a complete show stopper for us (as it seems to be for some) so we are watching to see where this ends up.

Illumina must be making a good faith effort to fix this and have just not found a solution yet. If they decide to take this SKU off the market (if it can't be fixed then I don't know what else they can do) that can only lead to more unhappy users than there currently are.

[dannyhi321]

This has not been a complete show stopper for us (as it seems to be for some) so we are watching to see where this ends up.

Illumina must be making a good faith effort to fix this and have just not found a solution yet. If they decide to take this SKU off the market (if it can't be fixed then I don't know what else they can do) that can only lead to more unhappy users than there currently are.

With all due respect, I don't regard the statement "There will be no public announcement until we have 100% identified the problem and removed it" relating to a known problem with a core reagent as acting in good faith. In fact a reagent whose performance metric many users base/based the purchase of their system. Keeping quiet and waiting until a user complains and then reimbursing them is hardly "acting in good faith".

I am glad car manufacturers do not apply the same "logic" to their safety systems!

[lac302]

Is this only an issue for amplicon sequencing? I haven't run a V3 600 cycle kit in a few months but have a library to sequence next week...WGS, FastQ only.

My last run worked fine even overclustered at 1800k/mm2, 85%PF.

I typically run the 600 cycle kits at 2x275. Quality over quantity is fine for my needs.