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  • Primer3 primer3_core failed

    Hi, I select 11 microsatellites generated by MISA program (http://pgrc.ipk-gatersleben.de/misa/), now I want to design primer, and I use this.

    Code:
    misa.pl FASTA.fa
    p3_in.pl FASTA.fa.misa
    primer3_core < FASTA.fa.p3in > FASTA.fa.p3out
    p3_out.pl FASTA.fa.p3out FASTA.fa.misa
    But the outgoing message is

    Code:
    11 record was created.
    Use of uninitialized value $count in concatenation (.) or string at ./p3_out.pl line 76, <SRC> chunk 11.
    Primer modelling was succefull for sequences.
    Primer modelling failed for 11 sequences.
    I chek the *.misa file, all is OK, but the problem is with primer3_core (I suppose) with the message "Use of uninitialized value $count in concatenation (.) or string at ./p3_out.pl line 76" and in the end with p3_out.pl the *.result show the SSR, SSR type but not the primers sequences.

    PD: I want to use the program and not the online service because after I have to do the same proces but with a genome and selec more than 100 microsatellites.
    Last edited by Daniel Fernandez; 02-21-2016, 07:43 AM. Reason: SOLVED

  • #2
    Originally posted by Daniel Fernandez View Post
    I chek the *.misa file, all is OK, but the problem is with primer3_core (I suppose) with the message "Use of uninitialized value $count in concatenation (.) or string at ./p3_out.pl line 76" and in the end with p3_out.pl the *.result show the SSR, SSR type but not the primers sequences.
    primer3_core is a compiled C program, it does not have variables like $count. The error message is definitely emitted by one of your Perl programs. Please run the 4 commands in an interactive shell, one after each other. Then you will see, which command throws the error.

    Comment


    • #3
      Originally posted by piet View Post
      primer3_core is a compiled C program, it does not have variables like $count. The error message is definitely emitted by one of your Perl programs. Please run the 4 commands in an interactive shell, one after each other. Then you will see, which command throws the error.
      Yes you're right, the problem is in ./p3_out.pl FASTA.fa.p3out FASTA.fa.misa

      It show me this:

      Code:
      Use for uninitialized value $count in concatenation (.) or string at ./p3_out.pl line 76, <SRC> chunk 11.
      Primer modelling was succesfull for sequences.
      Primer modelling failed for 11 sequences.
      What can I do?

      Comment


      • #4
        Start looking at FASTA.fa.p3out FASTA.fa.misa files to make sure that they are not empty or have some other issues in terms of format/content.

        Comment


        • #5
          The *.misa file is Ok:

          Code:
          ID	SSR nr.	SSR type	SSR	size	start	end
          Seq	1	p2	(TA)7	14	8423	8436
          Seq	2	p2	(AC)8	16	21149	21164
          Seq	3	c	(AT)13(GT)10	46	21525	21570
          Seq	4	c	(GAAA)16g(AAGA)12aaaagaaagaaaa(AAAG)5aaaaa(AAGG)5	171	24719	24889
          Seq	5	p2	(AC)15	30	31040	31069
          Seq	6	c	(TG)20ta(TG)7	56	51867	51922
          Seq	7	p4	(ATAG)14	56	71616	71671
          Seq	8	p2	(AT)8	16	76892	76907
          Seq	9	c	(AAGA)16agaaagaagaagaagaagaagaaagaagaaagg(AAGA)21	181	84544	84724
          Seq	10	p5	(AAAAC)6	30	85025	85054
          Seq	11	p4	(GAAG)5	20	88643	88662
          The *.p3out is this (I remove the sequence to show) 11 times the same.

          Code:
          PRIMER_SEQUENCE_ID=Seq_1
          SEQUENCE=AAAT ...CATC
          PRIMER_PRODUCT_SIZE_RANGE=100-280
          TARGET=8420,20
          PRIMER_MAX_END_STABILITY=250
          =
          And the *p3out is this (show the error) 11 times the same.

          Code:
          PRIMER_SEQUENCE_ID=Seq_1
          SEQUENCE=AAAT ...CATC
          PRIMER_PRODUCT_SIZE_RANGE=100-280
          TARGET=8420,20
          PRIMER_MAX_END_STABILITY=250
          PRIMER_ERROR=Missing SEQUENCE tag
          =

          Comment


          • #6
            Originally posted by Daniel Fernandez View Post
            Code:
            PRIMER_ERROR=Missing SEQUENCE tag
            =
            That means that primer3_core is complaining about missing SEQUENCE in its input file. Due to this, it has not found any primers. Then 'p3_out.pl' reads the output and cannot handle the case, that primer3 has not found any primers.

            Google-ing the primary error message "PRIMER_ERROR=Missing SEQUENCE tag" leads to a report of another user who has run into the same error in 2009, http://generic-model-organism-system...-td457437.html

            It seems that your version of primer3 is too new for your Perl scripts. You can fix this by simply replacing "SEQUENCE=" by "SEQUENCE_TEMPLATE=" in the primer3 input file ('FASTA.fa.p3in').

            Code:
            sed 's/^SEQUENCE=/SEQUENCE_TEMPLATE=/' FASTA.fa.p3in | primer3_core > FASTA.fa.p3out
            Last edited by piet; 02-18-2016, 09:29 AM.

            Comment


            • #7
              yes! now when I run primer3_core the *.p3out show me the primers:

              But when I run p3_out.pl the *.result file doesn't show me the complete table with the primers:

              Code:
              ./p3_out.pl FASTA.fa.p3out FASTA.fa.misa
              Use of uninitialized value $count in concatenation (.) or string at ./p3_out.pl line 76, <SRC> chunk 11.
              
              Primer modelling was successful for  sequences.
              Primer modelling failed for 11 sequences.
              The *.p3out file now is OK:
              Code:
              PRIMER_SEQUENCE_ID=Sequence_1
              SEQUENCE_TEMPLATE=AAAT...
              PRIMER_PRODUCT_SIZE_RANGE=100-280
              TARGET=8420,20
              PRIMER_MAX_END_STABILITY=250
              PRIMER_LEFT_NUM_RETURNED=5
              PRIMER_RIGHT_NUM_RETURNED=5
              PRIMER_INTERNAL_NUM_RETURNED=0
              PRIMER_PAIR_NUM_RETURNED=5
              PRIMER_PAIR_0_PENALTY=0.056998
              PRIMER_LEFT_0_PENALTY=0.030195
              PRIMER_RIGHT_0_PENALTY=0.026803
              PRIMER_LEFT_0_SEQUENCE=TCCTCATCCCAGCCCATGTA
              PRIMER_RIGHT_0_SEQUENCE=GCGGATTGATGCTCGTTGAC
              PRIMER_LEFT_0=88310,20
              PRIMER_RIGHT_0=88413,20
              PRIMER_LEFT_0_TM=60.030
              PRIMER_RIGHT_0_TM=59.973
              PRIMER_LEFT_0_GC_PERCENT=55.000
              PRIMER_RIGHT_0_GC_PERCENT=55.000
              PRIMER_LEFT_0_SELF_ANY_TH=0.00
              PRIMER_RIGHT_0_SELF_ANY_TH=0.00
              PRIMER_LEFT_0_SELF_END_TH=0.00
              PRIMER_RIGHT_0_SELF_END_TH=0.00
              PRIMER_LEFT_0_HAIRPIN_TH=0.00
              PRIMER_RIGHT_0_HAIRPIN_TH=0.00
              PRIMER_LEFT_0_END_STABILITY=2.2900
              PRIMER_RIGHT_0_END_STABILITY=3.1800
              PRIMER_PAIR_0_COMPL_ANY_TH=0.00
              PRIMER_PAIR_0_COMPL_END_TH=0.00
              PRIMER_PAIR_0_PRODUCT_SIZE=104
              PRIMER_PAIR_1_PENALTY=0.057388
              PRIMER_LEFT_1_PENALTY=0.030195
              PRIMER_RIGHT_1_PENALTY=0.027194
              PRIMER_LEFT_1_SEQUENCE=TCCTCATCCCAGCCCATGTA
              PRIMER_RIGHT_1_SEQUENCE=CTGCGGATTGATGCTCGTTG
              PRIMER_LEFT_1=88310,20
              PRIMER_RIGHT_1=88415,20
              PRIMER_LEFT_1_TM=60.030
              PRIMER_RIGHT_1_TM=59.973
              PRIMER_LEFT_1_GC_PERCENT=55.000
              PRIMER_RIGHT_1_GC_PERCENT=55.000
              PRIMER_LEFT_1_SELF_ANY_TH=0.00
              PRIMER_RIGHT_1_SELF_ANY_TH=0.00
              PRIMER_LEFT_1_SELF_END_TH=0.00
              PRIMER_RIGHT_1_SELF_END_TH=0.00
              PRIMER_LEFT_1_HAIRPIN_TH=0.00
              PRIMER_RIGHT_1_HAIRPIN_TH=0.00
              PRIMER_LEFT_1_END_STABILITY=2.2900
              PRIMER_RIGHT_1_END_STABILITY=4.1000
              PRIMER_PAIR_1_COMPL_ANY_TH=0.00
              PRIMER_PAIR_1_COMPL_END_TH=0.00
              PRIMER_PAIR_1_PRODUCT_SIZE=106
              PRIMER_PAIR_2_PENALTY=0.061380
              PRIMER_LEFT_2_PENALTY=0.034186
              PRIMER_RIGHT_2_PENALTY=0.027194
              PRIMER_LEFT_2_SEQUENCE=TCCCAGCCCATGTAAGCAAG
              PRIMER_RIGHT_2_SEQUENCE=CTGCGGATTGATGCTCGTTG
              PRIMER_LEFT_2=88316,20
              PRIMER_RIGHT_2=88415,20
              PRIMER_LEFT_2_TM=60.034
              PRIMER_RIGHT_2_TM=59.973
              PRIMER_LEFT_2_GC_PERCENT=55.000
              PRIMER_RIGHT_2_GC_PERCENT=55.000
              PRIMER_LEFT_2_SELF_ANY_TH=0.00
              PRIMER_RIGHT_2_SELF_ANY_TH=0.00
              PRIMER_LEFT_2_SELF_END_TH=0.00
              PRIMER_RIGHT_2_SELF_END_TH=0.00
              PRIMER_LEFT_2_HAIRPIN_TH=0.00
              PRIMER_RIGHT_2_HAIRPIN_TH=0.00
              PRIMER_LEFT_2_END_STABILITY=4.0100
              PRIMER_RIGHT_2_END_STABILITY=4.1000
              PRIMER_PAIR_2_COMPL_ANY_TH=0.00
              PRIMER_PAIR_2_COMPL_END_TH=0.00
              PRIMER_PAIR_2_PRODUCT_SIZE=100
              PRIMER_PAIR_3_PENALTY=0.061419
              PRIMER_LEFT_3_PENALTY=0.026803
              PRIMER_RIGHT_3_PENALTY=0.034616
              PRIMER_LEFT_3_SEQUENCE=GTCAACGAGCATCAATCCGC
              PRIMER_RIGHT_3_SEQUENCE=AGAGTGTTGCAGGCTCTCAC
              PRIMER_LEFT_3=88394,20
              PRIMER_RIGHT_3=88563,20
              PRIMER_LEFT_3_TM=59.973
              PRIMER_RIGHT_3_TM=59.965
              PRIMER_LEFT_3_GC_PERCENT=55.000
              PRIMER_RIGHT_3_GC_PERCENT=55.000
              PRIMER_LEFT_3_SELF_ANY_TH=0.00
              PRIMER_RIGHT_3_SELF_ANY_TH=18.19
              PRIMER_LEFT_3_SELF_END_TH=0.00
              PRIMER_RIGHT_3_SELF_END_TH=9.32
              PRIMER_LEFT_3_HAIRPIN_TH=0.00
              PRIMER_RIGHT_3_HAIRPIN_TH=34.86
              PRIMER_LEFT_3_END_STABILITY=5.5400
              PRIMER_RIGHT_3_END_STABILITY=3.5100
              PRIMER_PAIR_3_COMPL_ANY_TH=0.00
              PRIMER_PAIR_3_COMPL_END_TH=0.00
              PRIMER_PAIR_3_PRODUCT_SIZE=170
              PRIMER_PAIR_4_PENALTY=0.061810
              PRIMER_LEFT_4_PENALTY=0.027194
              PRIMER_RIGHT_4_PENALTY=0.034616
              PRIMER_LEFT_4_SEQUENCE=CAACGAGCATCAATCCGCAG
              PRIMER_RIGHT_4_SEQUENCE=AGAGTGTTGCAGGCTCTCAC
              PRIMER_LEFT_4=88396,20
              PRIMER_RIGHT_4=88563,20
              PRIMER_LEFT_4_TM=59.973
              PRIMER_RIGHT_4_TM=59.965
              PRIMER_LEFT_4_GC_PERCENT=55.000
              PRIMER_RIGHT_4_GC_PERCENT=55.000
              PRIMER_LEFT_4_SELF_ANY_TH=0.00
              PRIMER_RIGHT_4_SELF_ANY_TH=18.19
              PRIMER_LEFT_4_SELF_END_TH=0.00
              PRIMER_RIGHT_4_SELF_END_TH=9.32
              PRIMER_LEFT_4_HAIRPIN_TH=0.00
              PRIMER_RIGHT_4_HAIRPIN_TH=34.86
              PRIMER_LEFT_4_END_STABILITY=5.1800
              PRIMER_RIGHT_4_END_STABILITY=3.5100
              PRIMER_PAIR_4_COMPL_ANY_TH=3.02
              PRIMER_PAIR_4_COMPL_END_TH=0.00
              PRIMER_PAIR_4_PRODUCT_SIZE=168
              =

              Comment


              • #8
                Try to change the tag 'SEQUENCE_TEMPLATE' back to 'SEQUENCE' before you run 'p3_out.pl'. Maybe the missing 'SEQUENCE' causes the failure of 'p3_out.pl'.

                Code:
                sed 's/^SEQUENCE_TEMPLATE=/SEQUENCE=/' FASTA.fa.p3out > FASTA.fa.p3out2
                p3_out.pl FASTA.fa.p3out2 FASTA.fa.misa

                Comment


                • #9
                  I did that, but it show me the same

                  Code:
                  Use of uninitialized value $count in concatenation (.) or string at ./p3_out.pl line 76, <SRC> chunk 11.
                  
                  Primer modelling was successful for  sequences.
                  Primer modelling failed for 11 sequences.

                  Comment


                  • #10
                    Maybe more tags have changed name with the newer versions. Try an older version of primer3, 0.9 definitely shows the old behaviour. Tarballs are still available for download.

                    Last edited by piet; 02-20-2016, 02:49 AM.

                    Comment


                    • #11
                      I try to install it in my laptop but in the process it show me error, I used biolinux in my lab, it have installed by default.
                      Well with the *.p3out I extract the primers and other parameters with egrep command.
                      Thank you very much Piet.

                      If someone has the same problem

                      misa.pl FASTA.fa
                      p3_in.pl FASTA.fa.misa

                      # replace SEQUENCE by SEQUENCE_TEMPLATE
                      sed 's/^SEQUENCE=/SEQUENCE_TEMPLATE=/' FASTA.fa.p3in > FASTA.fa.p3in2

                      primer3_core < FASTA.fa.p3in2 > FASTA.fa.p3out

                      # It should work
                      sed 's/^SEQUENCE_TEMPLATE=/SEQUENCE=/' FASTA.fa.p3out > FASTA.fa.p3out2
                      p3_out.pl FASTA.fa.p3out2 FASTA.fa.misa

                      # If the above step doesn't show results correctly try this
                      egrep "PRIMER_LEFT_0_SEQUENCE" FASTA.fa.p3out
                      # the same with this words
                      PRIMER_RIGHT_0_SEQUENCE
                      PRIMER_LEFT_0_TM
                      PRIMER_RIGHT_0_TM
                      PRIMER_PAIR_0_PRODUCT_SIZE

                      Comment


                      • #12
                        Originally posted by Daniel Fernandez View Post
                        I try to install it in my laptop but in the process it show me error, I used biolinux in my lab, it have installed by default.
                        Well with the *.p3out I extract the primers and other parameters with egrep command.
                        Thank you very much Piet.

                        If someone has the same problem
                        Hi Daniel, did you already solved the problem on:
                        "Primer modelling was successful for sequences.
                        Primer modelling failed for 11 sequences."

                        I have the same issue, just wanted to ask if you already found a workaround.

                        Thanks

                        Comment


                        • #13
                          p3_out.pl never worked, I did manually with egrep, with this I can solve.

                          Comment


                          • #14
                            Hi Daniel, do yo know how to perform the same command (egrep) using command prompt in windows?

                            Comment


                            • #15
                              Originally posted by cdseq View Post
                              Hi Daniel, do yo know how to perform the same command (egrep) using command prompt in windows?
                              Sorry I don't work on windows, but I search and I think is findstr, I never test this. look these links.


                              Any recommendations on grep tools for Windows? Ideally ones that could leverage 64-bit OS. I'm aware of Cygwin, of course, and have also found PowerGREP, but I'm wondering if there are any hidden ...

                              Is there a similar utility to grep available from the Windows Command Prompt, or is there a third party tool for it?

                              Is there a command prompt grep equivalent for Windows 7? That is, I want to filter out the results of a command: Bash use: ls | grep root What would it be from a Windows command prompt?

                              Comment

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