Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Tapestation quality peaks show unwanted larger fragments.

    Hi all,

    I used Covaris to shear human genomic DNA at a set size of 300bp and 200bp respectively. This is to sequence my samples using NEB library prep kit on a MiSeq platform.

    I have however noticed, although both versions of Covaris programs set seem to work, I have also a rather significant portion of my DNA quality peak larger than 200 and 300bp as initially expected. The 200bp peak extends to 700bp whilst the 300bp extends to 1000bp. (File attached)

    My questions are:
    1. Is this ok to proceed for sequencing? How will it effect my overall coverage and sequence quality?
    2. If i decide to fine tune my protocol for size selection, will i lose important sequence information?
    3. I am not a big fan of Tagmentation using Transposase (which is why I prefer Covaris). However, say I use Tagmentation method to shear my DNA (and Nextera for Lib prep), will I see similar peaks? Is size fragments larger than desired ok for sequencing?
    4. What can i do to avoid getting the extended fragment peaks? In other words, how do i narrow the size of the peaks (without size selecting them)

    Any advice and help will be deeply appreciated. Thanks in advance
    Attached Files
    Last edited by mzahir89; 08-19-2015, 02:20 AM.

  • #2
    1- If they are your final libraries, a left side bead cut would help to remove fragments with small inserts. If they are sheared DNA you can do the left side cut prior to library prep step. Larger fragments will not affect sequencing if libraries are quantified correctly by qPCR.
    2- No, but library diversity (# of unique reads) is depend on input amount among other factors. Whether size select or not depend on availability of DNA and downstream application of data.
    3- Nextera library size distribution range will be wider than your sheared DNA.
    4- DNA can be sheared to shorter fragments which will result in narrower size distribution but then you need to adjust number of sequencing cycles to library’s average insert size.

    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