Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Amplification free library preparation

    Hello community,

    Firstly I would like to greet everyone, for this is my first of hopefully many productive experiences on this forum.

    My question involves amplification free library preparation for MiSeq. We have purified aptamers of 80 bp and would like to prepare them for sequencing with as little amplification as possible.

    Is it possible to directly ligate indexes to aptamers are do we have to ligate adapters first?

    Is there any other way?

    Thank you in advance for any suggestions.

  • #2
    The ligation of adapters (which contain your index) occurs right before amplification so as long as you have enough material/high enough library yield for clustering you don't necessarily need to amplify.

    Comment


    • #3
      Alternatively, you can use UMI - unique molecule identifiers. Each adapter that you ligate contains abarcode (of the library) and a short sequence (4-6 nt) unique for that molecule. After amplification you count each UMI of each DNA species only once, even if you got multiple reads of it and overcome amplification bias.
      Another amplification with less bias is IVT - in-vitro transcription which is linear rather than exponential as in PCR.

      Comment


      • #4
        Firstly, thank you for your help.

        After wavering few possibilities, we have decided that probably easiest and cost effective way is to use cloning kit to repair ends of the aptamers, create single d-ATP overhangs on 3' end and ligate adapters to them. Afterwards we made compromise and we will use standard Illumina index PCR to label.

        Another solution is KAPA hyper prep kit, that ligates adapters and indexes all together, but is a bit more expensive.

        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