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  • 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!

  • #2
    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

    Comment


    • #3
      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
      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

      Comment


      • #4
        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.

        Comment


        • #5
          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)?

          Comment


          • #6
            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!


            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 Files

            Comment


            • #7
              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.

              Comment


              • #8
                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...


                Originally posted by JoeKutch View Post
                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.

                Comment


                • #9
                  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.

                  Comment


                  • #10
                    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.

                    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 Files

                    Comment


                    • #11
                      %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.

                      Comment


                      • #12
                        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.

                        Comment


                        • #13
                          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 Files

                          Comment


                          • #14
                            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, 08:52 AM. Reason: Added additional questions

                            Comment


                            • #15
                              Jessica- Here's the thumbnails, metrics of a successful run.
                              Attached Files

                              Comment

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