Unconfigured Ad

Collapse
X
 
  • Time
  • Show
Clear All
new posts
  • JBKri
    Member
    • Jan 2014
    • 80

    #1

    Base composition vs cycle, MiSeq vs HiSeq X

    I have noticed that in our MiSeq runs we seems to always have an abrupt change in base composition in the last one or two cycles. The proportion of A goes to zero. Also I noticed we have a cyclic pattern in base proportions in the rest of the cycles, except the first 10 or so where there seems to be some other bias going on.
    On the other hand, the data from a HiSeq X run shows no such cyclic variation, nor the sudden drop in As at the end.
    See this image for base composition comparisons

    Does anyone have an idea of what is causing these patterns in the MiSeq, and why they are not seen in the HiSeq run?
    Note that these were two different libraries from different organisms. Both were TruSeq DNA PCR-free with Covaris ultrasonic shearing.
    Jon
    Last edited by JBKri; 06-20-2018, 06:02 AM.
  • JBKri
    Member
    • Jan 2014
    • 80

    #2
    On further investigation I see that the pattern varies depending on the number of cycles in the sequencing kit, and also some variation among runs. It seems 2*150 cycles causes little or no sudden change in base proportion in the last cycle. A few 2*250 runs showed some change in bases proportions, while the 2*300 cycle runs typically show the sharp change in the last cycle. Now the quetion is, should I trim off that last base? I have noticed the pattern can remain also after doing quality trimming.

    Comment

    • GenoMax
      Senior Member
      • Feb 2008
      • 7142

      #3
      You could run "n+1" cycles and then trim the last base, if you need that last base. You may want to chat with Illumina support to see if they have an explanation.

      Comment

      • moatman
        Junior Member
        • Dec 2016
        • 5

        #4
        Does this relate to the first base(s) of the adapter in your sample sheet? I've run into that problem before, where adapter trimming will eat the last little bit, even if it happens to just be the first base of the adapter, in this case maybe your missing A's.

        Comment

        Latest Articles

        Collapse

        • SEQadmin2
          16S/ITS Profiling vs. Shotgun Metagenomics: What Changes in Your Workflow?
          by SEQadmin2




          NGS has revolutionized clinical microbiology by offering new ways to identify and surveil pathogens without prior knowledge of the target. One popular approach is 16S rRNA and ITS amplicon sequencing, which targets specific microbial markers. But studies are increasingly incorporating shotgun metagenomics, a technique that sequences all DNA from a sample without targeting specific microbial markers. This approach allows more detailed taxonomic classification, detection...
          Today, 06:46 AM
        • SEQadmin2
          Multiomic Liquid Biopsy: What DNA/RNA Panels Reveal That DNA Alone Can’t
          by SEQadmin2



          Tumors contain complex mixtures of cell populations that evolve in response to treatment and host-specific factors. Tissue biopsies provide only static snapshots of a tumor’s state. But what if we could follow that evolution over time using a minimally invasive technique?

          Liquid biopsies can detect tumor-derived material circulating in the bloodstream, providing a way to study treatment response, detect emerging resistance, and discover new biomarkers. Circulating
          ...
          10-14-2026, 09:55 AM

        ad_right_rmr

        Collapse

        News

        Collapse

        Topics Statistics Last Post
        Started by SEQadmin2, 10-28-2026, 09:57 AM
        0 responses
        24 views
        0 reactions
        Last Post SEQadmin2  
        Started by SEQadmin2, 10-28-2026, 09:51 AM
        0 responses
        15 views
        0 reactions
        Last Post SEQadmin2  
        Started by SEQadmin2, 10-22-2026, 11:13 AM
        0 responses
        68 views
        0 reactions
        Last Post SEQadmin2  
        Started by SEQadmin2, 10-22-2026, 10:48 AM
        0 responses
        48 views
        0 reactions
        Last Post SEQadmin2  
        Working...