Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Can we stop the run and re-generated the cluster on flow cell on MiSeq?

    We are using the MiSeq machine (2*250, V2 kit) for a Truseq library sequencing. As we find the cluster density is low (only 239k) on the cycle 3, can we just stop the machine now and add additional libraries and re-run the protocol?

    Because the MiSeq machine used to report an error just after the cluster generation step. We washed and restarted the machine, and all process worked fine. I just wondering if I can do that again on cycle 3.

    add:
    The run continue and yield 2.7G result at last. The normal run with density 800K should yield 7.5G data.

    add:
    We quantified the libraries with real-time PCR using KAPA kit.
    Last edited by runsheng; 04-22-2015, 05:45 PM. Reason: add result

  • #2
    As far as I know, you cannot stop and restart the MiSeq. You should try to figure out why your cluster density is so low. How did you quantify your libraries before loading them on the MiSeq?

    Comment


    • #3
      Originally posted by microgirl123 View Post
      As far as I know, you cannot stop and restart the MiSeq. You should try to figure out why your cluster density is so low. How did you quantify your libraries before loading them on the MiSeq?
      We quantified the libraries with real-time PCR using KAPA kit. It works well for the RNA-seq libraries with insertion size ~500bp. As for the DNA library (insertion size ~800), the concentration should be lower than the calculated one, adjusted as 5/8.

      Comment


      • #4
        Interesting. Have you contacted Illumina tech support? The last time I saw such low cluster densities on a properly-quantified run, there was a problem with the optics.

        Comment


        • #5
          microgirl is right-- you can't just switch the instrument off and on and re-cluster a flow cell.

          The end of the cluster gen process includes a 3' blocking step that prevents extension of DNA template and flow cell oligos (which would interfere with bridge amplifying any new libraries you might add in an effort to boost cluster counts). The only work around I can think of would be to run a deblock step similar to what happens during the PE turnaround but I don't know how you would do that manually. You would still have to worry about having enough reagents to repeat clustering from the same reagent cartridge, though.

          Comment


          • #6
            Originally posted by microgirl123 View Post
            Interesting. Have you contacted Illumina tech support? The last time I saw such low cluster densities on a properly-quantified run, there was a problem with the optics.
            I guess the machine should be all right cause we just got a normal run for RNAseq one week ago.

            We have analysed the data generated from this run. Though with a low density and yield, the read seems to be fine and even get a little bit higher Q30 percentage than our former runs (with >800 cluster density). The quality, read length, read yield for each index and read mappbility all seem to be OK.

            The only flaw is the read coverage, we expected a 8X coverage but now we only get 3X.
            And that's why I asked if I can re-do the cluster generation process.

            Comment


            • #7
              Originally posted by Jessica_L View Post
              microgirl is right-- you can't just switch the instrument off and on and re-cluster a flow cell.

              The end of the cluster gen process includes a 3' blocking step that prevents extension of DNA template and flow cell oligos (which would interfere with bridge amplifying any new libraries you might add in an effort to boost cluster counts). The only work around I can think of would be to run a deblock step similar to what happens during the PE turnaround but I don't know how you would do that manually. You would still have to worry about having enough reagents to repeat clustering from the same reagent cartridge, though.
              Thank you for reply!
              Indeed we have re-generated the cluster once. At that time, we encountered an error after the cluster generation step and the machine was halted on cycle 1. After wash and restart, the run completed normally and yielded full data.

              The 3' blocking is still a ddNTP blocking for MiSeq?

              Comment


              • #8
                hi Runsheng,
                I understood you compared an 800 nt library qPCR result with a 500 nt library, and corrected loading with 5/8.
                This seems logical given both libraries cluster equally efficient.
                This is not the case. Shorter fragments cluster far more efficient.
                That is probably why you got a lower cluster density than expected.

                Comment

                Latest Articles

                Collapse

                • seqadmin
                  Strategies for Sequencing Challenging Samples
                  by seqadmin


                  Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                  03-22-2024, 06:39 AM
                • seqadmin
                  Techniques and Challenges in Conservation Genomics
                  by seqadmin



                  The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                  Avian Conservation
                  Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                  03-08-2024, 10:41 AM

                ad_right_rmr

                Collapse

                News

                Collapse

                Topics Statistics Last Post
                Started by seqadmin, Yesterday, 06:37 PM
                0 responses
                10 views
                0 likes
                Last Post seqadmin  
                Started by seqadmin, Yesterday, 06:07 PM
                0 responses
                10 views
                0 likes
                Last Post seqadmin  
                Started by seqadmin, 03-22-2024, 10:03 AM
                0 responses
                51 views
                0 likes
                Last Post seqadmin  
                Started by seqadmin, 03-21-2024, 07:32 AM
                0 responses
                67 views
                0 likes
                Last Post seqadmin  
                Working...
                X