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Old 07-18-2017, 01:29 PM   #1
Joanna
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Question Miseq v2- Low cluster density, Low PF%, what should we do?

Hi everyone,

We have been sequencing bacterial 16S rRNA gene libraires for about 3 years, prepared with custom dual-index primers (not staggered), followed Kozich paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753973/). But recently we have experienced some failures in sequencing these libraries, all showing very low cluster density and PF%. We generally load 4 pM (optimal based on Kozich paper), and spike in 15% PhiX. And our libraries were purified with AMPure beads twice and checked by running an agarose gel.

We have already re-ordered sequencing primers, re-made fresh dilutions of PCR primers and increased loading concentration to 8 pM, but cluster density was still not improved.

Has anybody experienced similar problems? Or can you please provide some advice? Thanks a lot!
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Old 07-19-2017, 03:14 AM   #2
nucacidhunter
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Possible causes:

1- low quality of sequencing or PCR primers resulting from synthesis or degradation during storage: in this case % of PhiX reads should be higher than expected
2- issues with sequencer or library denaturation: good indicator is lower than expected number of PhiX reads
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Old 07-19-2017, 06:14 AM   #3
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Hi nucacidhunter,

Thanks for your reply! We did get much higher PhiX than expected. We thought about low quality so we tried new dilutions from our stock primers and new sequencing primers. All did not work... Can you think of any other possible causes in this case?

Joanna
Quote:
Originally Posted by nucacidhunter View Post
Possible causes:

1- low quality of sequencing or PCR primers resulting from synthesis or degradation during storage: in this case % of PhiX reads should be higher than expected
2- issues with sequencer or library denaturation: good indicator is lower than expected number of PhiX reads
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Old 07-19-2017, 08:28 AM   #4
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If the sequencer appears to be working correctly and you've ruled out any issues with the primers, I think the library is the next best area to troubleshoot.

You mentioned you made new primer dilutions from stock. How are the remaining reagents-- buffers, enzymes, etc.? Are they new or do you have other cause to believe them to be contaminant free? The first thing I throw away and replace in cases like this is the PCR water.

Lastly, how are you quantifying your final library? Are you using a PCR-based method like the KAPA kit to make sure the DNA has adapters ligated in the correct orientation? It seems unlikely you'd have any issues if the P5 and P7 oligos are part of the PCR primers, but I've found this is always worth asking.

I'm sorry in advance since these are all pretty basic suggestions, and you may have checked most of these things out already.
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Old 07-19-2017, 08:33 AM   #5
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Also, can you post some screenshots of the run metrics: the run and lane metrics table, an intensity vs cycle plot (the charts dashboard page from basespace is perfect), and maybe a screenshot of one of the thumbnail images (assuming you save them)?
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Old 07-19-2017, 10:04 AM   #6
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Hi Jessica,

Thanks a lot for your advice.
We did change the PCR water and buffer etc. at the first place to make sure there's no contamination. And our primer has i5 and i7 adapters in it, so we just quantified with bio-analyzer and qPCR.

I am attaching the run metrics table and intensity/Q30 vs cycle plot, as well as the first thumbnail image for this run. Please let me know if you need any else information. Thanks for your help!


Quote:
Originally Posted by Jessica_L View Post
Also, can you post some screenshots of the run metrics: the run and lane metrics table, an intensity vs cycle plot (the charts dashboard page from basespace is perfect), and maybe a screenshot of one of the thumbnail images (assuming you save them)?
Attached Images
File Type: jpg s_1_1101_a.jpg (55.8 KB, 22 views)
Attached Files
File Type: pdf Q30 vs cycle.pdf (137.8 KB, 32 views)
File Type: pdf intensity vs cycle.pdf (287.7 KB, 29 views)
File Type: pdf run metrics.pdf (178.7 KB, 31 views)
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Old 07-19-2017, 10:23 AM   #7
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Looking at your run metrics, I think your phasing/prephasing seems high, which could indicate a clog/leak in your machine's fluidics. Our MiSeq had a similar problem a while back where we were only getting partial template and reagent delivery to the flow cell due to a pin-hole sized leak in the fluidics system.

Edit: That probably would not explain the disparity between your expected PhiX reads and the observed amount, though.
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Old 07-19-2017, 11:38 AM   #8
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Hi JoeKutch,

Thanks for the comment. I need clarify that we don't have the sequencer. We are working with the sequencing center in our university. They have done the troubleshooting with Illumina on their end and said that the sequencer was working fine. So now we are thinking about other possibilities...


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Looking at your run metrics, I think your phasing/prephasing seems high, which could indicate a clog/leak in your machine's fluidics. Our MiSeq had a similar problem a while back where we were only getting partial template and reagent delivery to the flow cell due to a pin-hole sized leak in the fluidics system.

Edit: That probably would not explain the disparity between your expected PhiX reads and the observed amount, though.
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Old 07-19-2017, 05:36 PM   #9
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I wonder if you could post %Base vs Cycle plot and library profile if you have or average expected amplicon fragment size.

I still think that the issue is PCR primer or sequencing primer. You have mentioned that you have used fresh dilution of stock but stock could have been degraded since you have used it last time and concentration or sequence of your sequencing primer reported by manufacturer could be inaccurate. I would suggest to check the primers on polyacrylamide gel or small RNA Chip.

Any mismatch in critical positions between adapter and sequencing primer could result in failure.
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Old 07-19-2017, 06:03 PM   #10
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Yes I agree we can't exclude the possibility of degradation of our stock primers. We probably still need check that.

I am also attaching the %base vs cycle and DNA assay result. We targeted V3-V4 region so our amplicon fragment size was ~500bp.

Quote:
Originally Posted by nucacidhunter View Post
I wonder if you could post %Base vs Cycle plot and library profile if you have or average expected amplicon fragment size.

I still think that the issue is PCR primer or sequencing primer. You have mentioned that you have used fresh dilution of stock but stock could have been degraded since you have used it last time and concentration or sequence of your sequencing primer reported by manufacturer could be inaccurate. I would suggest to check the primers on polyacrylamide gel or small RNA Chip.

Any mismatch in critical positions between adapter and sequencing primer could result in failure.
Attached Images
File Type: png DNA assay.png (49.2 KB, 13 views)
Attached Files
File Type: pdf %base vs cycle.pdf (317.5 KB, 25 views)
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Old 07-19-2017, 11:34 PM   #11
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%Base plot looks fine. There is large quality drop after ~cycle 150 that I thought might be due to primer-dimers in the library but does not seem to be the case, though it is difficult to know from the BA trace due to overloading.
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Old 07-20-2017, 07:47 AM   #12
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I noticed the phasing/prephasing numbers, too, Joe, but when compared to test 16s data from Illumina on basespace, the data here really don't seem all that extreme.

Joanna-- you said you quantify with qPCR before you load? I assume the concentrations were okay this time? Did you say in your OP that you've had more than one run do this? Have your yields have been consistent from prep to prep since you started making 16s libraries? Nothing has changed in your methodology recently? Has anything changed at the sequencing core? They don't have a new guy who's forgotten to use your custom primers, right? (I've seen it happen before, which is why I ask.)

I wonder if you could post one more thing-- for the tile you posted above, can you post the C,G,T images, as well? There's a lot of shadowing on the A image and I want to make sure the other images all similar. I expect it's nothing but I'd like to look, all the same.

I'm starting to lean in the direction of nucacidhunter that your sequencing primers might be the culprit.
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Old 07-20-2017, 08:14 AM   #13
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Jessica- All the QC and sequencing were done by the sequencing core. And based on their comments, everything looked fine for them. And I think we had a good communication about using custom primers.

This is actually the third run that I did and probably ~10th run in our lab. We followed the same protocol all the time and generally it had been working well in spite of some run to run variance. But one thing that we slightly changed for this run was the %PhiX. For the first two runs I did successfully, we spiked in 25% and 20% of PhiX respectively (we thought our samples might have very low sequences diversity). And then we felt it might be unnecessarily too much so decreased to 15% for this run. Do you think this could be the problem here?

I am attaching the thumbnails of other bases: C, T, G.
Attached Images
File Type: jpg s_1_1101_c.jpg (53.8 KB, 14 views)
File Type: jpg s_1_1101_t.jpg (54.1 KB, 17 views)
File Type: jpg s_1_1101_g.jpg (59.0 KB, 13 views)
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Old 07-20-2017, 09:50 AM   #14
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I'm not sure. The change in PhiX might be the issue-- this does seem to be pretty low diversity sequence, but I can't imagine that 15% instead of 20% could make that much of a difference.

I'm a little concerned about the haziness that surrounds the clusters in the T image, but since 70% of the identified clusters are PhiX, I'm not sure that it relates to low sequence diversity of the 16s libraries. I've sometimes seen sequencers do things like this when the cluster density is very low. Cutting the amount of PhiX that got loaded may have not provided enough fluorescent signal for the sequencer? You might try going back up to the 20-25% you were using previously and see if that helps at all.

Would you be able to attach the four thumbnails for an image from the previous run that worked well? I wonder if the haziness is an issue that came up this time and caused the run to fail. Could you share the run metrics, too?

Lastly, I assume temperature and humidity are stable in the core lab?

Last edited by Jessica_L; 07-20-2017 at 09:52 AM. Reason: Added additional questions
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Old 07-20-2017, 11:09 AM   #15
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Jessica- Here's the thumbnails, metrics of a successful run.
Attached Images
File Type: jpg s_1_1101_t.jpg (57.1 KB, 14 views)
File Type: jpg s_1_1101_g.jpg (56.1 KB, 8 views)
File Type: jpg s_1_1101_c.jpg (60.2 KB, 7 views)
File Type: jpg s_1_1101_a.jpg (56.4 KB, 7 views)
Attached Files
File Type: pdf run_metrics.pdf (828.3 KB, 8 views)
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Old 07-20-2017, 12:21 PM   #16
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It looks to me like you might be having two problems, though the degree to which each contributes to a failed run may be hard to isolate

The 16s libraries seem to be causing an issue since they're rather low complexity. The first 25 cycles or so want a decent signal in each channel-- the first five cycles for cluster registration and the remaining twenty for calculating the PF rate. MiSeqs assume a more balanced % base composition for the purposes of calculating dye crosstalk, too, so if you have extremely weak signal (which you do in A and G) the system will miscount and/or fail more clusters than it otherwise should.

You should be able to address this problem by bringing up the amount of PhiX that you load on the flow cell-- even if 70% of the aligned sequence on this run is PhiX, there's not enough signal in the A and G channels, I think. That 70% is 70% of 13% X 330K, which is a cluster density of something like 30K PhiX/mm^2. Maybe other users can provide information on their experiences, but I think you need more clusters than that to get good data. You said you're using a spike in of 20-25% Phix, so I assume that is in reference to the 4pM library which means you might be using a lot less PhiX (1-2pM?) than Illumina recommends for runs with low complexity libraries. For a low complexity library, you might want to be more in the 5pM range for PhiX.


Also, it appears as though you are having some kind of issue with library hybridization to the flow cell or sequencing primer hybridization, which is why your cluster density on this last run is so low in comparison to the other run you shared (the most recent run cluster count is three times lower). The fact that you reloaded these libraries at a concentration of 8pM and saw little change in cluster density is proof of this. If you saw a higher raw cluster count but a similarly low PF rate, then I think we could conclude that the PhiX is the primary problem, but if the raw cluster count is similarly depressed when loading more library, I think you have several things to look at, two of which are nucacidhunter's suggestions: the PCR primers/adapters and the sequencing primers.

The next suggestions may be more relevant to the sequencing core you use: if you've ruled out the oligos and the libraries passed QC at the core, and assuming the lab quantifies the libraries via qPCR, it might be worth checking out the NaOH that's used to denature libraries. NaOH does go bad, especially in the presence of CO2 from the air. Libraries should be denatured with a fresh NaOH dilution (I'm in the habit of checking the pH of my dilutions with pH strips to ensure it's above 12). Also, stock solutions should be double checked to make sure the math works out-if the core lab recently ordered new NaOH stocks and got 10N instead of 1N, the calculations in the Illumina denature/dilute guide are going to be off by a factor of 10. Carryover of excess NaOH results in incomplete neutralization with the hyb buffer, which can then interfere with flow cell hybridization.

I apologize for the wall of text, but hopefully these suggestions will be of some help to you.

Last edited by Jessica_L; 07-20-2017 at 12:23 PM. Reason: Edited for clarity.
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Old 07-20-2017, 12:48 PM   #17
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Jessica- Thanks so much for your super clear and logical explanation! I really appreciate your patience and expertise.

I think your comments on low complexity and uneven signals for each base really makes sense. I also thought about this possibility but didn't know how to explain this problem in details. And I just checked that when we loaded 8pM for the same libraries, cluster density indeed increased a bit (from 350 to 462) and the %PF was still as low (~17%). I think your conclusion is correct. Our primary problem is probably low complexity of the libraries and primers might also be less efficient than before.

Thanks again for your help!
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Old 07-21-2017, 02:46 AM   #18
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Detected cluster density is affected by following:
1- Loading: sometimes this can be off due to quantification errors
2- Library denaturation which is affected by incubation time and pH of NaOH
3- pH of hybridisation buffer-library pool
4- Sequencing primer hybridisation and extension which is dependent on level of primer sequence complementarity to adapter
5- Cluster density
6- Other factors

Summary table indicates 7M detected clusters/reads which around 1M has passed the filter and from that 700k is PhiX (10%). This indicates that around 5% of amplicon library reads has passed the filter. %Base vs cycle also shows relatively good diversity for 16S (does not show any 0 or 100 values for any base in any cycle). These are evidence of poor sequencing priming caused by primer (PCR or sequencing or both). There could have been even more clusters that has not been detected because they have not been primed and extended to generate a signal.
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Old 07-21-2017, 07:30 AM   #19
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No problem! Keep us posted on your progress and let us know if you need any more help.
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Old 07-21-2017, 07:41 AM   #20
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I run mostly 16s libraries as well. 15% 8pM phiX should be enough. I only quibit my libraries so not as accurate as kapa, but I load 8pm library. Illumina tech support has said to try not to go below 750k clusters or the software has a hard time assigning basecalls.
I also use double the recommended primers because they were designed before Illumina released their exact cycling specs and are on the outside of the temp range. I've had to have my temp control module replaced-it wasn't holding temp well but was apparently close enough for the phiX or illumina libraries to work but the 16s to fail.
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