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  • pmiguel
    replied
    Hi Mariana,
    Sorry, I haven't logged in since March. Looks like maybe you have extremely low complexity bases. You would want to cut your loading concentration way down -- like to 5-6pM and add at least 30% high complexity library -- like phiX.
    Regards,
    Phillip

    Leave a comment:


  • MarianaDS
    replied
    pmiguel thanks for answering. I have a very weird problem, perhaps you have encountered something similar in the past?

    We are developing a custom UMI-based method and hence we made our own forked adapters (i7: 8 nt + UMI: 25 random N); i5: 8 nt). We do paired-end seq in a Miseq with a 300 reagent kit (it has enough reagents for 329 cycles). So we use 140 cycles for R1 and R2, 32 cycles for i7 and 8 cycles for i5). We do not need to use custom primers.
    We start with randomly fragmented dsDNA from amplicons (~300 bp). Before PCR, we dilute our template to have a limited number of molecules and obtain higher family sizes; hence we expect libraries with low diversity. The PCR works fine, the tapestation profile looks nice (no adapter dimers shown). We loaded 13 pMol of our pool in a Nano flow cell (multiplexed 8 libraries) + 10% PhiX as spike-in. After a few hours we saw a high cluster density and the run finished without errors; however we obtained no yield, no %PF, no PhiX aligned and no fastq files.
    My interpretation:
    • Looking at the thumbnails images (attached in the post); I see that in all cycles from R1 underclustering (o perhaps background noise only?)
    • R2 ( index i7) seems to have clusters, at some cycles we see overexposure, which is in line with having too few molecules before PCR => too low diversity specially in the index region
    • R3 (index i5) looks similar to i7
    • in R4 interestingly we do see nice clusters
    • Altogether, I am suspecting that the libraries are able to bind to the flow cell, the primer binding regions for index i7 (and R4) is okay, but the region corresponding to Read1 priming site went missing. Perhaps our homemade forked adapters did not annealed properly?
    • What I can not explain, is why the heck we do not see PhiX aligned... I came across here a post about a "Deadly" library that would "kill" everything else in the flow cell, even PhiX. But the reason is not explained.
    By the way, I thought that the the Cluster Density was determined based on Read 1, so I do not understand why when looking at all the thumbnails and seeing almost no clusters we get to that huge number.
    We run in a weekly basis a Illumina prep kit and there we obtain beautiful cluster density + good data. The device has also been checked by a Field Engineer and the optics seem to be fine.

    Any help or idea is appreciated
    Our next step is to clone that library and pick few colonies & sequence them with Sanger to see what is inside P5 and P7.

    Mariana

    Leave a comment:


  • pmiguel
    replied
    Yeah, anything clustered at 15 pM on a v2 cassette is going to fail to produce PF clusters. I think you can conclude from Kyle's lack of a response to my diagnosis of his issue, that was the issue.
    That concentration is too high. The MiSeq software attempts to do an initial count of the clusters but can't count most of them because they are too close together or just confluent. So you just get the number of clusters it could identify in your average cluster density. But even these are too close to other clusters and fail chastity checks.
    That said, tile images should be produced.
    Of course this is just me re-diagnosing Kyle's issue. You don't mention what concentration you clustered at.

    Leave a comment:


  • MarianaDS
    replied
    Originally posted by KyleWolf View Post
    Hello everyone.

    Lurked for a while, but first time posting. I have a bit of a vexing issue.

    We are using a MiSeq Micro kit (v2 chemistry, 300 cycles).

    Libraries were mixed, denatured, PhiX added, loaded on the cartridge, etc.
    (15pM loading, 5% PhiX @ 10pM)

    The run started and it completed, but did not generate any data except cluster density (cluster density around 900, which I have already determined why it might be low).
    -no cluster diversity
    -no tile images
    -no % PF
    -no % aligned
    -no sequence data
    -no undetermined bins


    I don't believe it is a sample issue as the libraries were quantified using kapa, looked at via Tapestation, and re-quantified by Qbit for good measure (just to get library quality out of the way as a reason). and adjusting for a slight mistake calculating the library concentrations, the cluster density is what I would expect had the rest of the run work.

    PhiX had been previously denatured and used before and after this run with success.

    We have never used the Micro kit before and tech support had suggested there was nothing special you had to do or change in the sample sheet or LRM (since the kit's RFID code is read by the machine), but it is the only logical place that makes sense. This is based on the fact that the PhiX had worked on other runs that were using the Micro kit without issues, and for the % aligned to not show up with the PhiX makes me think there was a setting or something that made it so no data was collected.

    Has anyone ever seen an issue like this before? Could really use some help as I have been beating my head against my desk for the last 2 weeks.
    Hi Kyle,

    I was wondering whether you found out the reason for your issue. We are experiencing a similar problem in our lab..
    Any hints?


    Kind regards,
    Mariana


    Leave a comment:


  • pmiguel
    replied
    Originally posted by pmiguel View Post
    Are you getting the cluster density from SAV? If not, where?

    --
    Phillip
    I am asking because normally if I saw odd results from a run -- where there were no PF% calculated at all -- I would check the images using SAV. If there were no images, but the run finished on the instrument, then I would check the thumbnail images on the MiSeq itself.

    15pM seems like a crazy high clustering density to me. Since you included 5% phiX, it makes me think your libraries are low diversity? No way you could cluster a low diversity library at 15pM on a v2 cassette and get PF clusters. At least not in my experience. We usually cluster at 5pM (where library concentration is determined by KAPA qPCR) on v2 cassettes.

    So your story makes perfect sense if you clustered at 15pM and got zero % PF clusters. What seems weird is that you got no % PF reading at all. You should get something there, even if that something is "zero".

    --
    Phillip

    Leave a comment:


  • pmiguel
    replied
    Originally posted by KyleWolf View Post
    Hi Phillip,

    Thanks for the response! You are correct, there was no passing filter calculated. Same for alignment or cluster diversity, and no fastq data generated. Only the cluster density was generated.
    Are you getting the cluster density from SAV? If not, where?

    --
    Phillip

    Leave a comment:


  • KyleWolf
    replied
    Originally posted by pmiguel View Post
    By "no % PF", do you mean, PF% was zero, or PF% was not calculated? If the latter, then that is an issue independent of library quality. I have never seen this, but it sounds more like an issue with the computer.
    The micro and nano cassettes are identical to the standard cassettes. Near as I can tell, Illumina literally blacks-out part of the flowcell to "create" a micro or nano kit.
    --
    Phillip
    Hi Phillip,

    Thanks for the response! You are correct, there was no passing filter calculated. Same for alignment or cluster diversity, and no fastq data generated. Only the cluster density was generated.

    Leave a comment:


  • pmiguel
    replied
    By "no % PF", do you mean, PF% was zero, or PF% was not calculated? If the latter, then that is an issue independent of library quality. I have never seen this, but it sounds more like an issue with the computer.
    The micro and nano cassettes are identical to the standard cassettes. Near as I can tell, Illumina literally blacks-out part of the flowcell to "create" a micro or nano kit.
    --
    Phillip

    Leave a comment:


  • KyleWolf
    replied
    Originally posted by GenoMax View Post
    Have you had Illumina tech support a look at this run? They can do that remotely. Sounds like there may be a hardware/software issue (assuming you have libraries that will amplify by qPCR).
    We have worked with Illumina tech support. They say there is nothing wrong with the machine itself and said it is probably a bad library prep. But Illlumina loves saying that. It's always the library prep haha.

    However, I don't believe that it is. library showed up by qPCR, Qbit, and Tapestation, with the results we would expect.

    I am just wondering if anyone here has experience with the MiSeq Micro kit and if there is anything you have to do to run that kit vs the regular v2 or v3 kit.

    Leave a comment:


  • GenoMax
    replied
    Have you had Illumina tech support a look at this run? They can do that remotely. Sounds like there may be a hardware/software issue (assuming you have libraries that will amplify by qPCR).

    Leave a comment:


  • MiSeq Micro kit issue, no data except cluster density

    Hello everyone.

    Lurked for a while, but first time posting. I have a bit of a vexing issue.

    We are using a MiSeq Micro kit (v2 chemistry, 300 cycles).

    Libraries were mixed, denatured, PhiX added, loaded on the cartridge, etc.
    (15pM loading, 5% PhiX @ 10pM)

    The run started and it completed, but did not generate any data except cluster density (cluster density around 900, which I have already determined why it might be low).
    -no cluster diversity
    -no tile images
    -no % PF
    -no % aligned
    -no sequence data
    -no undetermined bins


    I don't believe it is a sample issue as the libraries were quantified using kapa, looked at via Tapestation, and re-quantified by Qbit for good measure (just to get library quality out of the way as a reason). and adjusting for a slight mistake calculating the library concentrations, the cluster density is what I would expect had the rest of the run work.

    PhiX had been previously denatured and used before and after this run with success.

    We have never used the Micro kit before and tech support had suggested there was nothing special you had to do or change in the sample sheet or LRM (since the kit's RFID code is read by the machine), but it is the only logical place that makes sense. This is based on the fact that the PhiX had worked on other runs that were using the Micro kit without issues, and for the % aligned to not show up with the PhiX makes me think there was a setting or something that made it so no data was collected.

    Has anyone ever seen an issue like this before? Could really use some help as I have been beating my head against my desk for the last 2 weeks.

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