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Old 11-12-2015, 10:23 AM   #41
Brian Bushnell
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Originally Posted by toneta2013 View Post
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).
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Old 11-13-2015, 02:51 AM   #42
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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 We ran one library on out of date kit and got average Q30 at 82%, same library two days later on new kit- quality at 69%- so there is an effect, but we still got enough data.most 500 cycle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.
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Old 11-13-2015, 07:31 AM   #43
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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...

Last edited by toneta2013; 11-13-2015 at 07:38 AM.
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Old 11-13-2015, 07:42 AM   #44
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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!
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Old 12-29-2015, 03:05 AM   #45
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Default V2 kit is better than V3

Qscore dropping

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

Last edited by d00b; 12-29-2015 at 03:10 AM.
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Old 02-23-2016, 07:18 AM   #46
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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.
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Old 02-23-2016, 07:22 AM   #47
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Illumina have already stated they will not release a PQN for this issue, so don't expect that to change...
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Old 02-23-2016, 07:28 AM   #48
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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.
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Old 02-23-2016, 08:27 AM   #49
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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/Wh...nce-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/...00-issues.html

https://www.researchgate.net/post/wh...wer_than_i5923
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Old 02-24-2016, 01:07 AM   #50
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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.....
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Old 03-04-2016, 08:05 AM   #51
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Lightbulb Use asymetric run mode with 600 cycle kits.

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Originally Posted by dfhdfh View Post
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.
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.
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Old 04-02-2016, 02:47 AM   #52
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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?
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Old 04-04-2016, 04:05 AM   #53
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Originally Posted by ksawatzki View Post
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?
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Old 04-04-2016, 05:05 AM   #54
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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).
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Old 04-04-2016, 06:36 AM   #55
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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?
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Old 04-04-2016, 06:52 AM   #56
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@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.
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Old 04-04-2016, 06:56 AM   #57
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@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?
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Old 04-04-2016, 08:26 AM   #58
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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.
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Old 04-04-2016, 10:26 AM   #59
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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!
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Old 04-07-2016, 10:57 AM   #60
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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.
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