Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Running a 454 library in Ion torrent?

    Hi all,

    We have a shotgun library prepared for 454 and would like to run it in ion torrent for extra data. We learned that the sequencing adaptor A is shared, but that the other adaptor is not (P1 for torrent, B for 454). The B adapter was, however, used in torrent some time ago and its sequence seems to be still present in the be bead, following P1. So the questions are:

    - Should we convert by ligating just P1 adaptor?
    - Any chance that we could run with no additional library conversion?
    - Can an emulsion made with 454 primers and beads be loaded directly in torrent?

    Any input will be most appreciated.

  • #2
    Hi, The 454 LibL 'A' adapter is the same... but the LibA 'A' is not - you need to check this.

    once you have figured this out the easiest way to adapt the library is to design a 454 "B" to ion torrent P1 fusion primer and then PCR amplify your library (together with an 'A' primer) for an appropriate number of cycles (keep it as low as possible, say 10-15 cycles). Then you can use A/P1 primers to quant the library.

    We have converted a few libraries in this way.
    Good luck!

    Comment


    • #3
      Just to add to above (or amend) I think 10-15 cycles is prob over kill. If you have an established library already, even 6 cycles will yield 97% new ends. You can start with 5-10ng and 6 cycles will give plenty of product.

      Comment


      • #4
        Thanks both Bunce and JeremyDay for the input.

        We were trying to avoid additional PCR cycles, but this sounds like the most promising strategy. We are indeed using 454 LilL A adapter so we'll give it a try ...

        We learned that wells in torrent are smaller, so 454 beads are anticipated to work.

        Comment


        • #5
          I've done something similar as well. In my case, however, it was Lib-A library that I converted, so I used two different primers. I made one that used part of the Lib-A A adapter to switch it to the IT-A adapter, and did the same thing at the other end to switch the B to P1. There was still part of both of the old adapters left, but that didn't cause a problem.

          You just need to be careful of your library size. The One Touch is more sensitive to library size than the 454 emPCR chemistry is, and if the fragments are too big you won't get anything.

          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, 03-27-2024, 06:37 PM
          0 responses
          12 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-27-2024, 06:07 PM
          0 responses
          11 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          53 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          68 views
          0 likes
          Last Post seqadmin  
          Working...
          X