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  • #16
    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, 11:23 AM. Reason: Edited for clarity.

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    • #17
      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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      • #18
        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 hybridization buffer-library pool
        4- Sequencing primer hybridization 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).

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        • #19
          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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          • #20
            No problem! Keep us posted on your progress and let us know if you need any more help.

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            • #21
              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.
              Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

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              • #22
                Hi guys,

                I want to post some updates. As people suggested, we ordered fresh primers and made new libraries. Then loaded 6pM with 30% PhiX spike in. And we also doubled the volume of sequencing primers.

                This time we had a cluster density of 854 K/mm2 which has improved but PF% was still very low (48.78%) and yield was also low. Aligned PhiX% was just 15% instead of 30%.

                My thought is:
                Based on the attached thumbnail images, I feel it was over-clustered this time. And since we sequenced low diversity 16S rRNA gene libraries so we had uneven signal distribution for each cycle. This may cause trouble for the camera to correctly identify clusters. So we saw an overall drop in Q30% and "bleeding" (see attached). But this couldn't explain the low PhiX% than expected. And I also noticed that the Q30% of R2 (the first index read) was especially low. Wondering if others met this before? What do you think is the problem?

                Thanks!
                Attached Files

                Comment


                • #23
                  Joanna, looks to me as though you've resolved the library issue with the new primers. I would agree that this particular run is over-clustered. We observe similar quality issues when 16S libraries are clustered at this density. Reduce the cluster density and the Q30% will improve. We target cluster density of 650-750 K/mm2 with a 15% spike-in which works well for us on a MiSeq run. I've provided two attachments that display typical results.
                  Attached Files

                  Comment


                  • #24
                    lower than expected phiX indicates that your quant was off. If you loaded your library to get 30% and only could align 15% phiX, that means that your 16S was approximately twice as concentrated as you thought so took up twice as many spots on the flowcell as you planned. Recovering roughly the amount of phiX as you put on is a decent indication that your library quant was correct
                    Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

                    Comment

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