Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • adapter ligation and library amplification

    Hello all! I was discussing Ion Torrent library preparation (Ion Plus Fragment Library Kit) with a colleague and noticed that I might have previously misunderstood some things about the process. After reading up a little, am I understanding this correctly?

    During library preparation both A and P1 adapters are ligated to the amplicons. Since this is random the ratio of correctly formed A--fragment--P1 combinations and incorrect ones (A--fragment--A or P1--fragment--P1) is basically 50/50. This 50% ratio of undesired products stays the same through library amplification because all combinations are amplified equally. The difference is only in the emulsion PCR step because the incorrect adapter combinations won't be amplified here and will (hopefully) fall away during the template enrichment process.

    Previously I'd thought the library amplification step would reduce the ratio of undesired combinations, but that would only be true when using tailed adapters like with Illumina or some other strand-specific selection technique.

  • #2
    Not quite: supposedly, the combinations will not be amplified equally. Fragments that ligated the same A or P1 ds-adapter on both ends will have reverse-complementary ends. So after denaturation, while temperature is lowering to Tann, these single-stranded, adaptor-ligated DNAs are prone to form a hairpin that can impair amplification. So "A-f-A" o "P1-f-P1" molecules should amplify with a lower efficiency than "A-f-P1" fragments.
    Last edited by r.rosati; 11-05-2016, 05:55 PM.

    Comment


    • #3
      So neither I nor my colleague were completely right. Thanks for the answer and the link!

      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
      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