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  • Mis-priming of amplicons during emPCR

    Hi guys.

    I've been having problems recently with the Roche/454 amplification primers mis-priming my amplicon templates in regions with some weak complimentarity. This is really messing up my data by creating abortive, short DNA fragments on my beads.

    I'm trying to contact Roche about this, but I'd love to get a sense is this is something other people who are doing Lib-A sequencing have seen before. Since I've seen these emPCR artifacts occur three times in the last month I'm assuming it can't be an isolated problem here. The more instances of I can show of this problem perhaps the more support Roche/454 will be willing to provide.

  • #2
    I saw a lot of mispriming, but in different application, which results in large increase in number of reads with no key. Although Roche do not release the content of their Amplification Primer mix included in emPCR kit (I use Lib-L), I presume these are just regular well known emPCR primers, which do not include the key sequence, so reads with no key should come from mispriming. I think a feasible explanation is partial evaporation of water during emPCR resulting in increase of Mg concentration. Roche does not recommend use of sealing film, but I noticed that cap strips recommended with PCR plates come off after PCR rather easily, suggesting possible lost of tight seal during PCR. I use BioRad optical film and sturdy twin-tech plates, which allow me tighten the heated lid quite well.

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    • #3
      Originally posted by proteasome View Post
      I've been having problems recently with the Roche/454 amplification primers mis-priming my amplicon templates in regions with some weak complimentarity.
      I was wondering how you know this is happening? I mean what observations led you to this diagnosis?

      I ask because I find the GS-FLX run results particularly inscrutable. So if you have pierced that veil maybe you would share what you have seen?

      --
      Phillip

      Comment


      • #4
        Philip,

        I am not sure I understood the questions. I get this information form GS run browser, which shows how many reads passed filters. "No key" filter is the first one applied to all reads, so one can see how many reads were rejected as having no key sequence. In my case I noticed this when I was doing Nimblegen capture. The protocol calls for amplification of enriched library using standard emPCR primers. Being notoriously non-compliant, I sequenced captured library before and after re-amplification and was surprised to see a big qualitative differences of what was supposed to be the same library. While post-capture library has significant fraction of reads rejected as short, post-amplification library had a startling increase in number of reads rejected by the key filter. Later, I saw a large number of no-key rejections with libraries which were not subject of capture. Again, all this is discernible from inspection of run data in Run Browser. Unfortunately, Run Browser is installed only on PC that operates the sequencer and cannot be installed on an offrig server. I think this limitation is rather silly on the part of those who created 454 software installer. So if you do not run instrument yourself or do not have access to the attendant PC, you really cannot inspect run data since you get only a SFF file that is already processed. May be you can ask for an account on the attendant PC and connect using VNC Viewer, then you can run Run Browser on your data.

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        • #5
          Originally posted by yaximik View Post
          Philip,

          I am not sure I understood the questions.
          I would say that you did. Your answer is very interesting.
          Originally posted by yaximik View Post
          Unfortunately, Run Browser is installed only on PC that operates the sequencer and cannot be installed on an offrig server.
          Not true. gsRunBrowser is part of the software you get from Roche. It runs fine on linux boxes. (At least Centos/Redhat and Debian work.) I always look over my runs using it on an off-rig server. Actually I run NX on my Win7 box, and log into a Debian linux box that runs the Roche software. NX compresses the way VNC would, but gives a bit better resolution.

          Actually when I tried my usual Xwindows client software, Xming, for gsRunBrowser, it is very sluggish. But it still works.

          --
          Phillip

          Comment


          • #6
            I guess you are talking about software distribution for the big boy. It is not the case for GS Jr I use. Installers on the disk set and in downloaded installation packages check, I guess, connection to the instument and install only analysis package on offrig. So in order to run gsRunBrowser I have to either use VNCviewer, or go to the attendant PC. Over VNC, both from Linux and from WinXP, even the full color mode works reasonably well.

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            • #7
              Ah, you mean the GS Jr...
              Yeah, VNC does heavy compression, so I am sure it would be fine under most circumstances.
              Irritating about the gsRunBrowser only working on the rig for GS Jr. My sympathies.

              --
              Phillip

              Comment


              • #8
                Originally posted by proteasome View Post
                Hi guys.

                I've been having problems recently with the Roche/454 amplification primers mis-priming my amplicon templates in regions with some weak complimentarity. This is really messing up my data by creating abortive, short DNA fragments on my beads.

                I'm trying to contact Roche about this, but I'd love to get a sense is this is something other people who are doing Lib-A sequencing have seen before. Since I've seen these emPCR artifacts occur three times in the last month I'm assuming it can't be an isolated problem here. The more instances of I can show of this problem perhaps the more support Roche/454 will be willing to provide.
                I don't think we're seeing the same problem, but certainly had huge data losses in December. We use Lib-A. Unacceptably high loss due to 'read too short quality'. GSSupport said small fragments in our libraries, which was NOT true. Cutting amplification primer by 2x helped enormously on one re-run; not so much on the next. Apparently if your signal strength is too HIGH, even on long fragments, the s/w thinks they are short reads and drops them. Already have our filters fine-tuned and using rCafie processing, so no way to recover more reads. Wish the company put as much time into improving amplicon sequencing as they seem to for shotgun. We may have to somehow test amplification primer lots ahead of time.

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