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GenoMax 01-19-2016 03:01 PM

Quote:

Originally Posted by melop (Post 187877)
Dear all. I'm an end user. Recently we got 2 HiSeq 4000 lanes from a local sequencing facility (150bp x 2 reads), PCR-free gDNA libraries made at the same facility. The quality looks quite bad, especially in read 2, only 60% and 70% of bases >Q30, which doesn't pass the Illumina specs. In this case, is it a common practice for the facility to redo the sequencing for us? We ask because it sounded like they don't want to redo it for us... How about the policy at your sequencing core?

Local policies at facilities differ and it is possible that your facility may not have a specific one for 4000 data due to the peculiarities you have been reading about in this thread.

That said, there is certainly enough reason to show your QC data to the facility and discuss your concerns/available options. If only your samples show this trend and other samples on the flowcell did not have a problem then that may be a reason why the facility may seem reluctant to redo your samples.

There is no harm (except a bit of additional work) for the facility to open a ticket with Illumina and have them look at the metrics of run in question. If Illumina provides replacement reagents (that is they determine that there is potential reagent/hardware issue) then you should be able to get your samples re-sequenced at no charge. If no free replacement kit is forthcoming then your options revert to getting the sample sequenced on a different sequencer (2500) at full and/or subsidized cost (since the facility made your libraries they have a responsibility to get you usable data).

melop 01-19-2016 03:13 PM

I see. But is that a common practice to let the end user bare the loss even if the facility prepares the library knowing that they are going on a HiSeq 4000? I understand that if Illumina agrees to replace the reagents then there will be no problem, but I will be surprised if that should become the end user's burden if such a performance issue is caused by inadequate library prep (say insert size not tight enough or negligence in adapter cleaning, suggested by previous posts). One problem is that this core does not have any written policy regarding quality, and their policies all seem to be post hoc .....

Quote:

Originally Posted by GenoMax (Post 187880)
Local policies at facilities differ and it is possible that your facility may not have a specific one for 4000 data due to the peculiarities you have been reading about in this thread.

That said, there is certainly enough reason to show your QC data to the facility and discuss your concerns/available options. If only your samples show this trend and other samples on the flowcell did not have a problem then that may be a reason why the facility may seem reluctant to redo your samples. There is no harm (except a bit of additional work) for the facility to open a ticket with Illumina and have them look at the metrics of run in question.

If Illumina provides replacement reagents (if they determine that there is potential reagent issue) then you should be able to get your samples re-sequenced at no charge. If no free replacement kit is forthcoming then your options revert to getting the sample sequenced on a different sequencer (2500) at your cost.


GenoMax 01-19-2016 03:31 PM

Quote:

Originally Posted by melop (Post 187881)
I see. But is that a common practice to let the end user bare the loss even if the facility prepares the library knowing that they are going on a HiSeq 4000? I understand that if Illumina agrees to replace the reagents then there will be no problem, but I will be surprised if that should become the end user's burden if such a performance issue is caused by inadequate library prep (say insert size not tight enough or negligence in adapter cleaning, suggested by previous posts). One problem is that this core does not have any written policy regarding quality, and their policies all seem to be post hoc .....

You are referring to two separate issues and they need to be addressed separately. Sounds like you are ok with the run metrics explanation.

If you have questions about the quality of the libraries that were prepared then ask the facility to share their library QC data with you. Ask them if they bead treated the libraries as recommended? If the libraries look suboptimal then they may need to clean them up and re-run your samples at no cost. Again things you should discuss in good faith with your facility.

There are current instances (e.g. MiSeq 2x300 kits) where dismal Q-scores seem to rule late in read 2 but that does not affect the actual sequence. That may apply in your case as well. You may have perfectly good sequence data that has a Q-score issue that people have been discussing in this thread.

Brian Bushnell 01-19-2016 07:34 PM

I encourage you to measure the quality-score accuracy of the runs, as per this thread (near the top of the first post), if you want hard data to show to your sequencing facility. As GenoMax mentioned, quality scores do not always correctly reflect accuracy - I've seen them both too high and too low.

luc 01-19-2016 09:15 PM

It is possible (if the library was correctly sized and the run was not overloaded; unfortunately the latter is hard to diagnose on the HS4000 ) that the problem is due to the reagents as discussed in this thread. If we spend some time with the Illumina tech support on the phone, Illumina will usually replace the reagents for us in these cases.

We would definitely re-run your libraries (as they were generated by the core), but is actually very difficult to assign responsibility/blame for failures on the HS4000 if only a single lane is run.


Quote:

Originally Posted by melop (Post 187877)
Dear all. I'm an end user. Recently we got 2 HiSeq 4000 lanes from a local sequencing facility (150bp x 2 reads), PCR-free gDNA libraries made at the same facility. The quality looks quite bad, especially in read 2, only 60% and 70% of bases >Q30, which doesn't pass the Illumina specs. In this case, is it a common practice for the facility to redo the sequencing for us? We ask because it sounded like they don't want to redo it for us... How about the policy at your sequencing core?


GW_OK 01-20-2016 06:44 AM

Quote:

Originally Posted by luc (Post 187896)
It is possible (if the library was correctly sized and the run was not overloaded; unfortunately the latter is hard to diagnose on the HS4000 ) that the problem is due to the reagents as discussed in this thread. If we spend some time with the Illumina tech support on the phone, Illumina will usually replace the reagents for us in these cases.

We would definitely re-run your libraries (as they were generated by the core), but is actually very difficult to assign responsibility/blame for failures on the HS4000 if only a single lane is run.

I completely agree.

I am puzzled, though, why any Core would not immediately have had the run replaced by Illumina and reloaded if it did not meet spec.

neurula 01-25-2016 04:52 PM

Just to add another voice, we noticed this on our validation runs at install in late Nov/December. Q30s dropping after 100 cycles of read 2, ending the run at ~40%. Error rates up above 7-8% as well. It was actually most obvious in the control lanes of PhiX that we ran. Seems like it must be a reagent issue, probably with enzyme or cleavage mix.
This thread just prompted me to email our FAS about it again. We didn't have a very satisfactory response from tech support, they said they hadn't seen enough 4K runs to determine what was "normal", but that our runs meet spec.
My PI said he's heard through the grapevine that this problem is widespread though - at Yale, Cornell, NYGC etc.

melop 01-27-2016 09:00 AM

Quote:

Originally Posted by Brian Bushnell (Post 187890)
I encourage you to measure the quality-score accuracy of the runs, as per this thread (near the top of the first post), if you want hard data to show to your sequencing facility. As GenoMax mentioned, quality scores do not always correctly reflect accuracy - I've seen them both too high and too low.

This looks interesting and definitely will be useful for downstream analyses.
However I feel that the statistics should be measured in the same way Illumina does, since Illumina guarantees 75% bases >Q30 based on their software output.

melop 01-27-2016 09:07 AM

Quote:

Originally Posted by luc (Post 187896)
It is possible (if the library was correctly sized and the run was not overloaded; unfortunately the latter is hard to diagnose on the HS4000 ) that the problem is due to the reagents as discussed in this thread. If we spend some time with the Illumina tech support on the phone, Illumina will usually replace the reagents for us in these cases.

We would definitely re-run your libraries (as they were generated by the core), but is actually very difficult to assign responsibility/blame for failures on the HS4000 if only a single lane is run.

So I have 2 lanes, both of which fall below 75% >Q30 (60% and 70%). As far as I know, the other 6 lanes are all borderline, right at 75% >Q30. So the average across the whole flowcell would still be below standard. So perhaps there's a combination of different problems. I'm not sure how successful will they be in negotiating with Illumina with this borderline case though.

melop 01-27-2016 09:15 AM

Quote:

Originally Posted by GW_OK (Post 187929)
I completely agree.

I am puzzled, though, why any Core would not immediately have had the run replaced by Illumina and reloaded if it did not meet spec.

We are puzzled too. Last time I heard from them the core told us that they will only replace the run if Illumina replaces reagents for them. They were vague about what to do if Illumina doesn't. That was why I asked to see if this is a common practice among the other cores.

GenoMax 01-27-2016 09:44 AM

@melop: It is still not clear if you have talked with the facility about your data/concerns and what the result of that conversation was? Do you know if they contacted Illumina tech support about this run?

Based on your post in #29 it seems that other 6 lanes on this flowcell were very close to acceptable spec (Illumina spec only specifies 75% bases > Q30 for 2 x 150bp, which seems to refer to entire sequence output from that flowcell).

It is certainly possible that Illumina refused to provide free replacements. As with any customer service issue the kind of response one gets (in this case free replacements) may entirely depend on the support person you reach.

Ultimately your result may be due the characteristic of the library. A re-run may produce a very similar result. Only option then would be to remake the library and try again.

melop 01-27-2016 10:20 AM

Quote:

Originally Posted by GenoMax (Post 188306)
@melop: It is still not clear if you have talked with the facility about your data/concerns and what the result of that conversation was? Do you know if they contacted Illumina tech support about this run?

Based on your post in #29 it seems that other 6 lanes on this flowcell were very close to acceptable spec (Illumina spec only specifies 75% bases > Q30 for 2 x 150bp, which seems to refer to entire sequence output from that flowcell).

It is certainly possible that Illumina refused to provide free replacements. As with any customer service issue the kind of response one gets (in this case free replacements) may entirely depend on the support person you reach.

Ultimately your result may be due the characteristic of the library. A re-run may produce a very similar result. Only option then would be to remake the library and try again.

Yes, they said they will rerun if illumina replaces the reagent for them. They did not say what happens if not. But from the message they seemed to imply that rerunning is contingent upon their success in the negotiations. They stopped replying to our emails after that.

GenoMax 01-27-2016 11:03 AM

That is unfortunate. If they don't intend to re-run your samples for free (due to not getting free replacements) then they should have informed you.

Since they made the libraries this is one of those tough situations (for them, in terms of cost) where they should at least do something to retain your business (either re-run the current libraries to see if the problem recurs or remake the libraries for free but then charge you for running them, only if the new data looks good).

NextGenSeq 01-28-2016 09:15 AM

Quote:

Originally Posted by melop (Post 187877)
Dear all. I'm an end user. Recently we got 2 HiSeq 4000 lanes from a local sequencing facility (150bp x 2 reads), PCR-free gDNA libraries made at the same facility. The quality looks quite bad, especially in read 2, only 60% and 70% of bases >Q30, which doesn't pass the Illumina specs. In this case, is it a common practice for the facility to redo the sequencing for us? We ask because it sounded like they don't want to redo it for us... How about the policy at your sequencing core?

Illumina will almost always send replacement reagents if you use a small amount of spike-in PhiX sequenced with your library AND the PhiX did not give good sequencing results. If the PhiX gives good sequencing results the problem is the library not the instrument. If both give poor results it is likely an instrument or reagent problem.

If the sequencing facility did not do this they are amateurs and you shouldn't use them again.


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