Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Miniseq problem help

    An assay that I have run on the miniseq previously is starting to give me problems and I am at a loss on what it could be. My problem on the surface looks like underloading. My cluster density is around 124 k/mm2 and phiX is around 2.5%. However, I ran an old library the previously gave me good numbers (i.e. 240K/mm2, 1% phix) has dropped to the poor numbers I first mentioned. I swapped out lots of the flow cell and the results recovered. However, the following day the numbers dropped again and even worse on the third day. My cluster density decreased from 240 to 174 to 109 k/mm2. I looked at the image of the clusters and looks heavily clustered and does not look like 109 k/mm2. The other metrics like pass filter and Q30 etc are fantastic, above 95%. The problem is that I lose depth at all of my targeted amplicons as a result of the underclustering. I tried new NaOH and TrisHCl to no avail. It seems that my library is somehow binding less efficiently and phix isnt as affected. On a side note, I have noticed some bubbles in the flow cell but I am not sure if this is normal and introduced during the post run wash cycle. Any ideas will be helpful! Thanks!

  • #2
    I think your library might be degraded. I elute my library in Tris Low-EDTA buffer. It works well for me.

    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
    9 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-22-2024, 10:03 AM
    0 responses
    50 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