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  • multiplex primer design software (is there any?)

    Hi guys,

    I'm wondering if there is any software for multiplex PCR primer designs.

    I know about Primer3 and that a lot of other software use Primer3 as the core engine.

    I'm wondering if there is any algorithm out there that does two additional things:
    1) checks primer specificity (against hg19 reference), and/or
    2) checks primer-primer interaction, and thus choose a pool of primers that minimizes primer-dimer formations.

    If you guys can point me anywhere, I'd really appreciate it.

    Thanks in advance.

  • #2
    Originally posted by lethalfang View Post
    Hi guys,

    I'm wondering if there is y software for multiplex PCR primer designs.

    I know about Primer3 and that a lot of other software use Primer3 as the core engine.

    I'm wondering if there is any algorithm out there that does two additional things:
    1) checks primer specificity (against hg19 reference), and/or
    2) checks primer-primer interaction, and thus choose a pool of primers that minimizes primer-dimer formations.

    If you guys can point me anywhere, I'd really appreciate it.

    Thanks in advance.
    We have one that we created. Is the amount of primers you need very large? How many primers do you need to place in one tube?

    Let me know. I cannot guarantee that we can run it for you but maybe.

    Comment


    • #3
      Originally posted by math_guy View Post
      We have one that we created. Is the amount of primers you need very large? How many primers do you need to place in one tube?

      Let me know. I cannot guarantee that we can run it for you but maybe.
      Around 100 pairs of primers I guess, or less.
      Is it published anywhere that I can look?

      Comment


      • #4
        Originally posted by lethalfang View Post
        Around 100 pairs of primers I guess, or less.
        Is it published anywhere that I can look?
        unfortunately I can't give you the software. 100 primers is no problem for us though. I can ask our guys to run it and if they say yes then we can run it and send you the results. but...you would have to test our set using another method.

        i know what you mean and there is really no awesome publicly available primer design software so most labs have to do it themselves as far as I know. we can run it for you but I don't want to be responsible if you have results that you were not expecting.

        I suppose you could ask again on this forum if any lab has commercial software that they would run for you as a favor but I sorta doubt anyone here would do that...but try anyhow..i said yes

        Comment


        • #5
          Testing out there. Say I have extracted the coding regions of all the splice variants of EGFR, and added 50 extra bases before and after each region for primer design purposes.
          I generated the .fasta file here:


          For each entry, RealSeq:50-185 means the target region really goes from the 50th base to the 185th base, the 50 bases before and after were added as paddings to design primers.

          If the software works, can it be licensed or something?

          Comment


          • #6
            have you checked e.g.

            http://biocompute.bmi.ac.cn/CZlab/mprimer/
            ...

            Comment


            • #7
              Originally posted by dietmar13 View Post
              Yes I have looked at it.
              I think it has good algorithm, and I'm doing some "experiments" with MPprimers, but it's also missing some important features, e.g.,
              1) It does not do tiling. It takes input of a .fasta file, and it will design one single primer pair for each sequence, without regard to the length or position of the target regions of interest, and
              2) It does not have a way to define what is the target of interest in a given sequence. As the .fasta file I have included here, I have a notation that states the region of interest (e.g., from position 50 to position 138). Primer3 has that option, but it's not included in MPprimer.

              I would like to work with the MPprimers developers if that's possible......

              Comment


              • #8
                MPprimer 1.4 looks pretty good

                Comment


                • #9
                  Hi there.
                  This might be a little late. I am building a program to facilitate our multiplex assays so was wondering what sort of features you were looking for in your 'multiplex-primer design program. I have had a look at some of the other online programs and wasn't particularly thrilled with some of their features. We will be launching our program soon online.
                  Thanks

                  Comment


                  • #10
                    Originally posted by athena7793 View Post
                    Hi there.
                    This might be a little late. I am building a program to facilitate our multiplex assays so was wondering what sort of features you were looking for in your 'multiplex-primer design program. I have had a look at some of the other online programs and wasn't particularly thrilled with some of their features. We will be launching our program soon online.
                    Thanks
                    I'm no longer in this space, so I don't have any stake in this. It's definitely a very difficult problem:

                    1) Being able to set range for the replicon size, i.e., distance of the two primers, between 250 bp to 350 bp for instance.
                    2) When designing back-to-back primers to cover a long region (i.e., tiling), be able to control the distance between the left primer of the succeeding replicon and the right primer of the preceding replicon.

                    This is the most difficult:
                    3/a) Choose the right primers when you have a choice, so the interaction between the primers are minimized in each pool of primers;
                    3/b) Separate primers into multiple pools if the interaction cannot be avoided, but minimize the number of pools.

                    Comment


                    • #11
                      updated on multiplex primer software

                      Originally posted by athena7793 View Post
                      Hi there.
                      This might be a little late. I am building a program to facilitate our multiplex assays so was wondering what sort of features you were looking for in your 'multiplex-primer design program. I have had a look at some of the other online programs and wasn't particularly thrilled with some of their features. We will be launching our program soon online.
                      Thanks
                      Hi. You said that you were working on a program for multiplex primer design back in August. Have you completed it yet, and is it available? I'm interested in testing software for highly multiplexed amplicon sequencing (e.g., Campbell et al. 2015, doi 10.1111/1755-0998.12357). I want to be able to develop the locus-specific primers, and also add the primer tails and test for compatibility of tailed primers.

                      Comment

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