Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Earth Microbiome Locus Primers

    Is anyone using 515F and 806R as locus primers for the Illumina two-step amplicon PCR library prep?

    I have one investigator using them following the entire Earth Microbiome protocol (so adding primer pads and linkers) and sequencing with custom primers. This works great on the MiSeq and clusters properly.

    Several other people have recently submitted samples prepared with the two-step PCR process (so no primer pads, etc.) and the sequencing just uses the Illumina primers in the MiSeq kit. These libraries are all over-clustering, even at loading concentrations of 2 pM.

    Is anyone else seeing results like this? I'm starting to wonder if the locus-primers are somehow causing a problem with the sequencing or possibly the libraries are not working properly with the KAPA quantification.

    I just started rerunning a library that was slightly over-clustered (4 pM loading concentration) at 2 pM, and the cluster density didn't decrease at all!

  • #2
    I have not used these exact primers but in my experience two step PCR libraries sequence just fine. Aim for cluster density at 550-750k with 20% PhiX. Read number and variation are similar with qPCR and dsDNA fluorophores quantifications. I would check the libraries to make sure that amplicons are clean with no primer dimers and double check calculations.

    Comment


    • #3
      We have run libraries containing amplicons from these primers, and I haven't seen any clustering issue related to them.

      From my experience, the clustering of amplicons on the MiSeq is not linear, so when a run is severely over-clustered, I generally dilute the pool to half its concentration and then load the same volume of the diluted pool (I hope that makes sense).

      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
      67 views
      0 likes
      Last Post seqadmin  
      Working...
      X