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Old 12-08-2015, 06:37 AM   #1
GW_OK
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Angry 3000/4000 read 2

Anybody here with a 3000/4000 seen poor quality read 2's lately? I have attached example plots from 4 previous runs below. It seems to be library agnostic.

I've been lead to understand that there is an escalating investigation internal to Illumina into possible chemistry issues. They're not comp-ing runs until lot numbers are given for the failed runs, I know that.

Just wondering how far the problem extends.
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File Type: jpg 151203_Bioo_PCR-free.jpg (68.7 KB, 52 views)
File Type: jpg 151105_Kapa_hyper.jpg (49.2 KB, 34 views)
File Type: jpg 150930_Kapa_hyper.jpg (49.9 KB, 25 views)
File Type: jpg 151022_Nextera.jpg (44.2 KB, 34 views)
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Old 12-08-2015, 01:17 PM   #2
Brian Bushnell
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That is consistent with my analysis of data from the HiSeq 4000 platform, provided to us by Illumina. The chemistry (as of a few months ago) was absolutely not up to 2x150bp runs IMO, and read 2 particularly had very low quality. I also analyzed some 2x100bp PhiX data that had decent quality for both read 1 and read 2. So, the platform may be adequate for 2x100bp runs.
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Old 12-09-2015, 05:47 AM   #3
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We have usual "read2-gate" (like the antenna-gate, bend-gate for new apple products) for a new Illumina technology?

On a serious note, @GW_OK what kind of libraries are represented by these 4 examples?

Last edited by GenoMax; 12-09-2015 at 06:24 AM.
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Old 12-09-2015, 08:15 AM   #4
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Libraries given above are (in no order) a mix of:
Kapa Hyper Prep, Bioo Scientific Nextflex PCR-free, Illumina Nextera

We've got a Bioo Scientific Nextflex flowcell running now. I'll update once it completes.

Illumina is sending FSE's to pull our entire optics module and check alignments next week. There is hope that it will be less-bad afterwards.
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Old 12-09-2015, 05:35 PM   #5
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Hi GWOK, thanks for starting this thread!

We are experiencing the same problems (as is another lab in the neighborhood). The quality of the PE150 bp reads was perfectly fine a few months ago. Illumina likley has reagent quality problems at the moment.
The quality scores are now dropping considerably faster than a few months ago.
The % at which the median Q30 score curve ends (e.g. at cycle 310) seems to be a good quantifier for the problems. The curve used to end at about 60%, now often under 20%, even under 10%.

Illumina tech support did inform us that there are "NO reagent quality problems" - because all reagents individually pass QC. However they are investigating if there is a similar phenomenon ( a "non-problem"?) as with the MiSeq reagents which generate reduced read qualities scores since more than half a year now ( http://seqanswers.com/forums/showthread.php?t=59558 ).

Please keep us updated.

Last edited by luc; 12-09-2015 at 05:38 PM.
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Old 12-09-2015, 06:55 PM   #6
Brian Bushnell
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Luc, which platform (and chemistry, and software) are you having trouble with?

FYI, our HiSeq 2000/2500 machines seem fine, which is one of the reasons I have recommended against "upgrading" to HS3000 or 4000 (from which I have never seen good data). As far as I know, our NextSeq is fine too, but our MiSeqs are outputting junk for 2x300bp amplicon runs.

Last edited by Brian Bushnell; 12-09-2015 at 07:03 PM.
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Old 12-10-2015, 06:18 AM   #7
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Latest run has finished. Two large Bioo NextFlex pools across respective halves of the flowcell.

Read 2 still looks weak. One thing of note is that the top surface looks better than the bottom. That being said neither surface's read 2 look nearly as good as read 1.

luc, thanks for posting. I'm glad I'm not the only one seeing this.

I've also included a Q30 plot from Illumina's "HiSeq 4000: TruSeq PCR-Free (NA12878)" publicly shared run on BaseSpace as an example of what I imagine good runs are supposed to look like.
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Old 12-10-2015, 10:48 AM   #8
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Hi Brian,

We have seen the problems on the HiSeq 4000 the last 6 weeks, and since quite a while on our Miseqs (we are underclustering considerably for these to get any usable data).



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Luc, which platform (and chemistry, and software) are you having trouble with?

FYI, our HiSeq 2000/2500 machines seem fine, which is one of the reasons I have recommended against "upgrading" to HS3000 or 4000 (from which I have never seen good data). As far as I know, our NextSeq is fine too, but our MiSeqs are outputting junk for 2x300bp amplicon runs.

Last edited by luc; 12-10-2015 at 05:13 PM.
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Old 12-18-2015, 08:56 AM   #9
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Update.
Our FSE has been on site all week working on realigning the optical path and running several tests on the fluidics, hoping that this will mitigate the issue. Based on what the factory people told him from data sent off we were ever so slightly off in some sort of image correction model. I view the instrument tweaking as dubious but have been told that this must be done first before the chemistry is brought into question.

We'll have our first test run on Monday. I'll upload performance once it completes.
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Old 01-04-2016, 05:30 AM   #10
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Update.
After running a mix of PhiX and other libraries Q30 plots are still not great but perhaps less terrible. Attached here are two plots showing a PhiX lane as well as a Nextera lane (PhiX is missing one of the two index reads).

While neither are bottoming out as before there's certainly a steep downward trend in read after the intensity jump. This run did "meet spec" for a 3k run (75% >Q30) but Illumina still wants to send out an "instrument expert" to have a look at our machine.
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Old 01-04-2016, 05:41 AM   #11
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Two more since then have given mixed results. The first was a Bioo NextFlex PCR-free and is perhaps one of the best runs we've had on this machine. The Q30 plot is attached (the one with black boxes).

After that run another, with a mix of library types, gave poorer performance. The Q30 plot for this is also attached, this time with blue boxes.
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Old 01-08-2016, 03:50 PM   #12
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Ours tend to look unfortunately like your last example - we have not seen significant differences between customer prepped libraries our own libraries and also PCR-free libraries.
At what concentration are you loading the PCR-free libraries and and how many clusters did you sequence?

Thanks!
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Two more since then have given mixed results. The first was a Bioo NextFlex PCR-free and is perhaps one of the best runs we've had on this machine. The Q30 plot is attached (the one with black boxes).

After that run another, with a mix of library types, gave poorer performance. The Q30 plot for this is also attached, this time with blue boxes.
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Old 01-11-2016, 08:48 AM   #13
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A couple more runs that don't meet spec.

Here's the first. It's two pools of NextFlex PCR-free on respective halves. Loaded at 150pM.
Q30 tanks towards the end:


We have a look at the %base and say "Ah Ha, sneaky adapter dimers slipping in!". Granted one should be able to clearly see adapter dimer traces on a Tapestation or Bioanalyzer but with PCR-free adapters things don't migrate properly and sizes are all off (we have tried denaturing and running it on a RNA tape, with some success).


Apparently the ExAmp and ordered flowcells just go bonkers with short libraries and that's what's killing the end of our reads, right?

So we take the two pools, give them a 0.8x bead cleanup (as opposed to our typical 0.9x) and load them back up again. We load at 100pM this time to really knock down any possibility of polyclonal wells.

The %base plot shows no more adapter dimers:


And read 1 Q30 looks pretty good relative to what you can expect on a 3k, but read 2 Q30 is an almost straight slope down:


I'm given to understand there's yet another lab out there that's having troubles with a 3k/4k. They've decided to not even bother going out to 150bp and just truncate the runs to 120bp. If that's the way this ends up going here I better not have to pay for those extra cycles.
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Old 01-11-2016, 07:34 PM   #14
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From what I've seen, you'll get much better results doing 2x100 runs in the first place, rather than truncating the 2x150 runs...
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Old 01-12-2016, 05:23 AM   #15
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Our initial 4000 run (2D x 151) was a mix of random samples from past with a lane of phiX. With the caveat that n=1 things do not look too bad. Still analyzing the data.
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Old 01-18-2016, 11:06 AM   #16
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Another run. Not as bad this time. Still not 2500-level good (for read 2) but not as terrible as before.

A few things we've learned thus far (empirically and from talking Illumina) regarding the 3k:

1) Anything short will take over the ordered flowcell with a vengeance. As best you can, DO NOT have library fragments <300bp (150bp insert). ALL adapter dimer must be gone. Do at least a 0.8x SPRI cleanup. This flowcell is much less forgiving than the 2500.

2) If you have libraries on the shorter side (200-300bp insert) it is better to OVER load than UNDER load.
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Old 01-18-2016, 11:57 AM   #17
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Perhaps this is the reason why HiSeq X was such a controlled release and for a specific application. Patterned flowcells generate a lot of data but the quality is good (in terms of duplicates) only when libraries fit a narrow/strict quality profile.
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Old 01-19-2016, 07:28 AM   #18
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Timely article from James Hadfield reiterating some of the points from GW_OK

http://core-genomics.blogspot.co.uk/...d-to-know.html
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Old 01-19-2016, 09:01 AM   #19
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Thanks for the link. The article does not present anything new (yet)- James just received his HS4000.
We have been very happy with it, the exception being that the qualities of the PE150 reads are no longer as good as they were last spring and summer. The other mentioned caveats can be dealt with; long insert libraries need to be run on the Hiseq 2500 of course.

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Timely article from James Hadfield reiterating some of the points from GW_OK

http://core-genomics.blogspot.co.uk/...d-to-know.html
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Old 01-19-2016, 01:16 PM   #20
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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?
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