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  • Consistent low MiSeq cluster density and PhiX alignment with high quality reads (PF%)

    Hi everyone,

    I am struggling to understand an issue I am having with sequencing on an Illumina MiSeq. I have had success in the past with this exact protocol at a different institution - with another machine and reagents etc.

    I am sequencing custom 20 pM ddRADseq libraries using a V3 600 cycle kit.
    My workflow is;
    library prep > pippin prep > AMPure > Tape station > Bioline library QC qPCR kit > dilute to 10 nM > side by side denature and dilute 10 nM library and PhiX to 20 pM > spike in 10% PhiX.

    On my last run I got low cluster densities (365K/mm2) and low PhiX alignment (0.91% - aiming for 10%) with good sequence quality (91.51% >=Q30). This seems to be the way each run goes.

    Any help would be appreciated

    Michael.

  • #2
    One possibility that would explain this is that your phiX and library are not denaturing properly. How old is your NaOH? I always denature with NaOH, and then heat-denature my final combined library/phiX dilution.

    Comment


    • #3
      Thanks for the reply. My 1N NaOH is about 8 months old now. I make up my working solution as I am denaturing. This issue occurred 1 day after making my stock and 8 months later on my second run.

      Comment


      • #4
        Following up on microgirl123's post, do you periodically check the pH of the NaOH? The stock should be high of course, but even the 0.2N should be 13 or so.

        The only other immediate thing I can think of for low cluster density is that you're carrying over too much NaOH into cluster generation, but that usually only happens if you're using too much OH or if there's an issue with the HT1 buffer. It could happen because of the pipettes if they're not calibrated or an o-ring has gone bad on one of them.

        Comment


        • #5
          I haven't been checking the pH. It seems like the best place to start.
          I do use calibrated pipettes, but I also have an addition dilution step that reduces the final concentration of NaOH to 0.4 mM. Does anyone have any thoughts on my process?

          For denaturing and dilution:
          - start with a 10 nM library and 0.2N NaOH (library: 10,000 pM, NaOH: 200 mM)
          - 1:1 dilution with 0.2N NaOH (library: 5000 pM, NaOH: 100 mM)
          - Add 990 uL HT1 (library: 50 pM, NaOH: 1 mM)
          - Dilute library to 20 pM = 240 uL library:360 uL HT1 (library: 20 pM, NaOH: 0.4 mM)

          Comment


          • #6
            The NaOH concentrations are about what they should be for each individual step of the process. The only thing I haven't seen before is starting with a 10nM library-- the standard protocols I've seen all recommend starting with 4nM. I'm not sure that makes a difference, but it might be worth trying. Starting with 4 can still get you to 20pM if you load at that concentration.


            If you need to get the library to a higher concentration, you can follow the procedure that ArcherDx uses in their protocols, which generate a 40pM denatured library:
            10uL of 4nM library + 10uL of 0.2N NaOH
            Incubate 5 minutes at RT, then add 10uL of 200mM Tris pH 7
            +970uL of HT1
            The final concentration is a 40pM library and ~2mM NaOH. Since the library is more concentrated at this step, you use less of it, and dilute to a final loading volume of 1000uL.

            I've used both the standard Illumina protocol and this one, and had no problems with the cluster generation part of things, but again, we start with 4nM.

            Good luck!

            Comment


            • #7
              is your cluster density the same top and bottom of the flow cell? if you have sig more reads on the bottom you may be carrying over etoh from the ampure cleanup
              Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

              Comment


              • #8
                Originally posted by thermophile View Post
                is your cluster density the same top and bottom of the flow cell? if you have sig more reads on the bottom you may be carrying over etoh from the ampure cleanup
                This is interesting; do you know the mechanism this causes this result?

                Comment


                • #9
                  Originally posted by thermophile View Post
                  is your cluster density the same top and bottom of the flow cell? if you have sig more reads on the bottom you may be carrying over etoh from the ampure cleanup
                  Thanks for the suggestion! My cluster density is essentially the same on the top and bottom surfaces.

                  Comment


                  • #10
                    Have you looked at the thumbnails? does it look like low clustering? What are you sequencing-super high or low GC?
                    Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

                    Comment


                    • #11
                      I've attached a pretty typical thumbnail from my latest run. This looks on the low side to me. I am sequencing plants. The species I am working on are about 30-40 % GC content (based on post-run fastQC reports).
                      Attached Files

                      Comment


                      • #12
                        I agree, that looks low. and your GC isn't going to cause a problem.

                        I've been checking the pH of my diluted NaOH with every run-just a drop on pH paper helps me know that my NaOH is ok
                        Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

                        Comment


                        • #13
                          Originally posted by mamor View Post
                          I haven't been checking the pH. It seems like the best place to start.
                          I do use calibrated pipettes, but I also have an addition dilution step that reduces the final concentration of NaOH to 0.4 mM. Does anyone have any thoughts on my process?

                          For denaturing and dilution:
                          - start with a 10 nM library and 0.2N NaOH (library: 10,000 pM, NaOH: 200 mM)
                          - 1:1 dilution with 0.2N NaOH (library: 5000 pM, NaOH: 100 mM)
                          - Add 990 uL HT1 (library: 50 pM, NaOH: 1 mM)
                          - Dilute library to 20 pM = 240 uL library:360 uL HT1 (library: 20 pM, NaOH: 0.4 mM)
                          Our workflow is a little bit different when we use the MiSeq. Usually it is the following:

                          1. Normalize all samples to 8 nM and pool them together.
                          2. Qubit the pool to see how far off it is from 8 nM and normalize to 4 nM.
                          3. Take 5 uL of 4 nM library + 5 uL of 0.2N NaOH. 5 minute incubation.
                          4. Add 990 uL of cold HT1 buffer to get to 20 pM.
                          5. Dilute 20 pM library to final loading concentration.
                          6. Add final library to a new tube with amount of desired PhiX. (i.e 540 uL of 16.5 pM library + 60 uL of 16.5 pM PhiX for 10% spike-in).

                          If you are worried about the final concentration of NaOH left, you can always add 5 uL of 200 mM Tris-HCl after the denaturation step and add 985 uL of HT1 instead of 990 uL.
                          Last edited by itstrieu; 08-23-2019, 01:10 PM.

                          Comment


                          • #14
                            what is the size of your lib ? 10% PhiX is too high. Based on the kit you used (v3, 600 cycles), I assume that the size of your lib is larger than PhiX lib, so relatively PhiX is easier to align to oligos on flowcell than your own lib, consequently you have less target lib in results. My suggestion is to reduce Phix to 1% or less. When you make and denature your 20 pM lib, just use the dilute of ~100-1000 pM. My stock NaOH is 1 N, I have used it for three years, still good.

                            Comment


                            • #15
                              Originally posted by kmcarr View Post
                              This is interesting; do you know the mechanism this causes this result?
                              our guess is etoh floating to the top of the flowcell? We had a number of runs with sig more clusters on the bottom than top which started when a new tech started cleaning the pools. I watched her working and realized that she wasn't fully drying the beads before resuspending. Once we corrected that, the runs improved and the top bottom discrepancy went away.
                              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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