Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • custom sequencing primers

    Hi,

    Does anyone use their own primers for sequencing on the GAII, i.e rather than the Illumina supplied primer? If so, what purification/modifications do you use? I'm wondering if, for example, any phosphorothioate bonds would be necessary... any thoughts appreciated!

    Thanks.

  • #2
    I don't think the sequencing primers are modified. The sequences are freely available from Illumina if you ask (they may even be posted here somewhere).

    Cheers,

    Scott.

    Comment


    • #3
      The sequencing primers are indeed unmodified. We order PAGE purified although I am not sure there is a requirement for this. If you want to know the sequences I would suggest contacting Illumina techsupport directly and asking for the latest release of their "sequence information for customers" pdf.
      This way there should be no confusion.
      James.

      Comment


      • #4
        a sequencing primer OTHER than the Illumina primer

        Hi !
        My first post here
        Originally posted by athos View Post
        Hi,

        Does anyone use their own primers for sequencing on the GAII, i.e rather than the Illumina supplied primer?
        Possibly the same question that you had in mind, but phrased differently.

        What I would like to know is whether it is possible, when you are doing amplicon sequencing, to use a primer WITH A SEQUENCE DIFFERENT from that used by Illumina.
        Let me explain. Imagine you have amplified a region, say the flanking sequences of a transposon, and made an Illumina library that has the Illumina primers at both ends. But you don't want to sequence the region immediately downstream of the primer, because you already know what it is. You want to start sequencing WITHIN the insert.
        Can you prime sequencing with an oligonucleotide that anneals to a common part of all your inserts, downstream of the adapter ?

        If yes, must you use the same sequencing primer for the whole chip, or can you use different sequencing primers in different lanes ?

        Final question: is it true that identification of the clusters is hampered when the first nucleotides of all (or most of) the sequences are identical ? This would be the case for example if you were sequencing through polyA-tails, or through a region that is common to all your inserts. I have heard that it is wise in this case to add 2 Ns right after the sequencing primer to make sure that sequences differ locally, allowing pixels to be grouped into clusters. True ?

        Comment


        • #5
          1. You can use custom primers for sequencing. The Tm should be similar to the standard primer.

          2. You can use different primers in different lanes of the flow cell.

          3. Biased nucleotide composition in the first few cycles (I believe this number varies with different software versions) adversely affects cluster calling.

          -Harold

          Comment


          • #6
            It is indeed true that low-diversity at the start of sequences will cause problems with cluster identification. See this thread.

            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