Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • NimbleGen SeqCap EZ Choice Library-preparation for the PGM

    Hello all.

    From a previous project, we have a 'NimbleGen SeqCap EZ Choice Library'-design that we would now like to use for a new project on our Ion Torrent PGM.
    I am very new to next-gen sequencing, but it looks like I should be able to prepare a library (fragmented to appropriate PGM-size, PGM-adapter ligated, nick translated, size selected, amplified, and purified) according to the PGM protocols and then do the capture following NimbleGens protocol, followed by template preparation and sequencing according to the PGM protocols. The main problem seems to be the differences in the oligos used, i.e. during the NimbleGen capture 'hybridization enhancing'-oligos are added to the mixture. Can anyone please explain to me in detail what these are, how they are designed and how I might create a set corresponding to the PGM-adapters used?

    Also, according to the NimbleGen protocol a ligation-mediated PCR is performed after the capture. Can't I just do a PGM-protocol amplification here and achieve the same or am I missing a point here?

    Any advice would be greatly appreciated. Thanks in advance.

    Best regards
    Christian

  • #2
    "Hyb enhancing" oligos are blocking oligos that anneal to the adapter sequences of your library, to prevent one properly captured library fragment (on-target) from pulling down another via annealing in the adapter regions. This dramatically increases your on-target rate.

    In my experience with SureSelect, you should get >>70% on target if your design is sound, leaving out the blocking Oligos will net ~40% on-target.

    There is a Nimblegen document (easily googleable, but if you any find it, I'll post it), that describes the design of the hyb enhancing oligos for Illumina. One can likely use the same principles to design blocking oligos for the IT adapters.

    Alternatively, call Nimblegen. I would be surprised if they didnt have a canned PGM recommendation.

    Good luck!

    Comment


    • #3
      Thanks for the quick answer and forgive my late reply.

      I didnt manage to find the mentioned paper but contacted NimbleGen directly. They confirmed that it should be possible to exchange their HE-oligos with PGM adapter-complementary oligos and achieve the same, so I will give it a try very soon :-)

      Comment


      • #4
        please update us with you results, cuz I'm planning to do the same.

        Comment


        • #5
          SeqCap EZ on the Ion Torrent

          Dear all,

          I just found this on the internet, didn't have the time to read it all through but it seems that the hyb enhancing oligos sequences are in there.

          Cheers,
          Attached Files

          Comment


          • #6
            Great, this is spot on!
            Thanks a lot!

            Do you know who created this?

            Best regards

            Comment


            • #7
              Originally posted by sokram View Post
              Dear all,

              I just found this on the internet, didn't have the time to read it all through but it seems that the hyb enhancing oligos sequences are in there.

              Cheers,
              Thanks for this information.

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