Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • PhiX spike-in issue

    Hi all,

    I have come across issues with the Illumina PhiX v3 in my project. I need to sequence my low diverse library on NextSeq 500. To balance out the diversity, I tried to spike-in 50% of the Illumina PhiX v3 control along with my library. But, the run wasn't good and I only managed to see a 9% alignment in my first run. At first, I thought it was resulted from the degradation of my PhiX. Thereby, I quantified my PhiX using picrogreen and the reading seems fine after conversion and the bioanalyzer showed no sign of degradation. I repeated my run the 2nd time. This time the Illumina FAS came over and supervised my library denaturation method to ensure I followed exactly the instructions recommended in the protocol, ie. library denaturation->library dilution to 1.8pM followed by PhiX denaturation->dilution of denatured PhiX to 1.8pm and, eventually, mixed both library and PhiX at 50:50 ratio. Everything was fine but the results of the 2nd run was still the same as in we couldn't achieved near 50% of PhiX spike-in. In addition, we observed a high error rate too. Does anyone have any suggestion on this issue? Thank you in advance for the guidance.

  • #2
    Three issues to consider:
    1- your library and PhiX
    2- sequencing chemistry
    3- sequencing hardware

    For correct diagnosis you need to provide some information from runs SAV metrics such as cluster density, PF%, % aligned to PhiX, Q30 scores

    Comment


    • #3
      Hi @nucacidhunter,

      Thank you for your reply. Please see below for the metrics. Thank you in advance for your guidance.
      Attached Files

      Comment


      • #4
        The issues that is apparent:

        1- Low cluster density that could be due to errors in quantification, denaturation or loading resulting in under loading or overloading (over clustering where several cluster are identified as a single cluster). Over clustering possibility can be checked by looking at images and also selecting intensity in Flow Cell Chart from analysis page which should show higher values (yellowish) in upper tiles in comparison to lower ones. In standard clustering lower tiles has higher density than the upper ones.

        2- PF% very low: indicates low diversity (as you have mentioned) that has not been improved by PhiX spike-in because PhiX% is not high enough (this can be checked by looking at %Base in Data By Cycle plot in analysis tab).

        3- High error rate: could be result of over clustering or poor sequencing primer compatibility (if custom primer was used)

        4- If you can try quantifying PhiX by qPCR

        I hope this and looking to similarities in second run helps you to identify the issue. If you need more help please post images from those analysis page plots.

        Comment


        • #5
          Thank you very much for your reply, @nucacidhunter. The Illumina FAS is currently checking through the run metrics. He is also suspecting that the library sample itself might be overclustered, resulting in the template generation to perform poorly and also exlaining on why the alignment didn't make sense. We had actually run the 3rd time, in which we lowered the library sample loading concentration from 1.8 to 1.5pM and it did improve the cluster density. But, the error rate was still high.

          We did quantify our PhiX and the concentration was fine. So I guess we could omit the possibility of the degraded PhiX being used. I have attached the plot of data by cycle for this run for your reference.

          Thank you
          Attached Files

          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
          8 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, Yesterday, 06:07 PM
          0 responses
          8 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          49 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          66 views
          0 likes
          Last Post seqadmin  
          Working...
          X