Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • #16
    It seems unlikely that LNA bases would be used - to my knowledge, LNA in the template inhibits most polymerases.
    I assume the phosphorothioate linkage helps predominantly preventing 3’ to 5’ exonuclease activity of the ligase - reducing adapter dimers.
    We too, had good experiences with the Bioo adapters though.

    Comment


    • #17

      Reduction of non-insert sequence reads by dimer eliminator LNA oligonucleotide for small RNA deep sequencing


      Here we describe a method for constructing small RNA libraries for high throughput sequencing in which we have made a significant improvement to commonly available standard protocols. We added a locked nucleic acid (LNA) oligonucleotide—named dimer eliminator—that is complementary to the adapter-dimer ligation products during the reverse transcription reaction. It reduces adapter-dimers, which often contaminate standard libraries and increase the number of non-insert sequence reads.

      http://www.biotechniques.com/Biotech...es-304403.html

      Tufts LNA Adapter Protocol

      Comment


      • #18
        Dear NextGenSeq, (apologies for dragging up an old post!)

        I have a DNA enrichment project where our inserts range from only 40bp and up. We'd like to get them all.

        We've just done a run and it is clear that we have sequenced a lot of adapter dimers (we expected that to be the case from the bioanalyser trace). My question is how applicable your strategy for dimer removal would be to standard Illumina DNA-DNA adapter dimer junctions? From the % base trace on MiSeq reporter it appears that most of the adapter junctions have gained an A (complementary to the overhanging T).

        i.e. the pure adapter dimer junction should be: GCTCTTCCGATC/GATCGGAAGAGC
        But we see (I think): GCTCTTCCGATCtaGATCGGAAGAGC

        Or rather: in the sequence trace we see: AGATCGGAAGAGC...

        And since the read primer ends: ...GCTCTTCCGATCT

        This presumably means that the overhanging T is present (for the read primer to work) and an A has been added upstream of the GATCGGAA...

        So...questions:
        Would an oligo that spans this junction inhibit PCR?
        What is the role of the LNA within this oligo?
        Last edited by M4TTN; 08-09-2016, 10:36 AM.

        Comment


        • #19
          Originally posted by M4TTN View Post
          i.e. the pure adapter dimer junction should be: GCTCTTCCGATC/GATCGGAAGAGC
          But we see (I think): GCTCTTCCGATCtaGATCGGAAGAGC

          Or rather: in the sequence trace we see: AGATCGGAAGAGC...

          And since the read primer ends: ...GCTCTTCCGATCT

          This presumably means that the overhanging T is present (for the read primer to work) and an A has been added upstream of the GATCGGAA...

          So...questions:
          Would an oligo that spans this junction inhibit PCR?
          What is the role of the LNA within this oligo?
          I think adapter-dimer formation is more complex. I would suggest analysing a TruSeq Universal and Index adapter for hetero-dimer formation for instance using OligoAnalyzer. You will see that there is more than one way for hetero-dimer formation with relatively low Delta Gs (they will anneal during annealing step) and each of those can be extended and then those products can form new versions of hetero-dimers in the following cycles.

          In your target range the best way for getting rid of dimers is preventing or reducing dimer formation by careful titration of adapters or using dimer-free adapter technology.

          PS. I have not seen any proof that Illumina uses LNA technology in their adapter oligos but LNA will increase the stability of annealed adapter oligos.
          Last edited by nucacidhunter; 08-09-2016, 10:22 PM. Reason: Added more info

          Comment

          Latest Articles

          Collapse

          • seqadmin
            Current Approaches to Protein Sequencing
            by seqadmin


            Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
            04-04-2024, 04:25 PM
          • 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

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by seqadmin, 04-11-2024, 12:08 PM
          0 responses
          14 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 04-10-2024, 10:19 PM
          0 responses
          19 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 04-10-2024, 09:21 AM
          0 responses
          16 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 04-04-2024, 09:00 AM
          0 responses
          43 views
          0 likes
          Last Post seqadmin  
          Working...
          X