Dear all,
I am trying to use PRIMER3 to design primers on the Linux command line. Following the manual, I prepared a file called PRIME with the Boulder-IO syntax:
SEQUENCE_ID=e6
SEQUENCE_TEMPLATE=ATGTTTCAGGACCCACAGGAGCGACCCATAAAGTTACCAGATTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGCTTT
TCGGGATTTATGCATAGTATATAGAGATGGGAATCCATATGCAGTGTGTGATAAATGTTTAAAGTTTTATTCTAAAATTAGTGAGTATAGATATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACA
AACCGTTGTGTGATTTGTTGATTAGGTGTATTAACTGTCAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGC
AGATCATCAAGAACACGTAGAGAAACCCAGCTGTAA
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=1
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=18
PRIMER_MIN_SIZE=15
PRIMER_MAX_SIZE=21
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_PRODUCT_SIZE_RANGE=75-100
PRIMER_EXPLAIN_FLAG=1
=
Launching primer3 with the suggested syntax of primer3_core < file I get:
$ primer3_core < PRIME
SEQUENCE_ID=e6
SEQUENCE_TEMPLATE=ATGTTTCAGGACCCACAGGAGCGACCCATAAAGTTACCAGATTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGCTTTTCGGGATTTATGCATAGTATATAGAGATGGGAATCCATATGCAGTGTGTGATAAATGTTTAAAGTTTTATTCTAAAATTAGTGAGTATAGATATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACAAACCGTTGTGTGATTTGTTGATTAGGTGTATTAACTGTCAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGCTGTAA
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=1
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=18
PRIMER_MIN_SIZE=15
PRIMER_MAX_SIZE=21
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_PRODUCT_SIZE_RANGE=75-100
PRIMER_EXPLAIN_FLAG=1
PRIMER_ERROR=Unrecognized PRIMER_TASK
=
primer3_core: Unrecognized PRIMER_TASK
I tried to remove the file by sending the variables as argument by enclosing the data in " but I got:
$ primer3_core "SEQUENCE_ID=e6
> SEQUENCE_TEMPLATE=ATGTTTCAGGACCCACAGGAGCGACCCATAAAGTTACCAGATTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGCTTTTCGGGATTTATGCATAGTATATAGAGATGGGAATCCATATGCAGTGTGTGATAAATGTTTAAAGTTTTATTCTAAAATTAGTGAGTATAGATATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACAAACCGTTGTGTGATTTGTTGATTAGGTGTATTAACTGTCAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGCTGTAA
> PRIMER_TASK=generic
> PRIMER_PICK_LEFT_PRIMER=1
> PRIMER_PICK_INTERNAL_OLIGO=1
> PRIMER_PICK_RIGHT_PRIMER=1
> PRIMER_OPT_SIZE=18
> PRIMER_MIN_SIZE=15
> PRIMER_MAX_SIZE=21
> PRIMER_MAX_NS_ACCEPTED=0
> PRIMER_PRODUCT_SIZE_RANGE=75-100
> PRIMER_EXPLAIN_FLAG=1
> =
> "
Error opening file SEQUENCE_ID=e6
SEQUENCE_TEMPLATE=ATGTTTCAGGACCCACAGGAGCGACCCATAAAGTTACCAGATTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGCTTTTCGGGATTTATGCATAGTATATAGAGATGGGAATCCATATGCAGTGTGTGATAAATGTTTAAAGTTTTATTCTAAAATTAGTGAGTATAGATATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACAAACCGTTGTGTGATTTGTTGATTAGGTGTATTAACTGTCAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGCTGTAA
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=1
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=18
PRIMER_MIN_SIZE=15
PRIMER_MAX_SIZE=21
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_PRODUCT_SIZE_RANGE=75-100
PRIMER_EXPLAIN_FLAG=1
=
The same is true if I add a \n at the end of each line.
My questions are:
how shall I write the file to feed primer3_core?
Is there a way to avoid the file and give the data as arguments to primer3_core?
Thnak you
Hello marongiu.luigi ,
check each line of your PRIME file that there are no whitespaces at the end.
primer3 tells you what its problem is:
PRIMER_ERROR=Unrecognized PRIMER_TASK
I could reproduce thhis with the file content you've posted above. It turned out that you have whitespace at the end of the line PRIMER_TASK=generic. After removing those the next error arises due to the same problem,
fin swimmer
Thank you, now it works for me as well. It was a very tricky bit. It there a way to pass the parameters directly without need of a file?
By the way, I still getting an error at the end of the computation:
PRIMER_ERROR=Input line with no '=': ; Input line with no '=': ; Input line with no '=': ; Final record not terminated by '='
=
primer3_core: Input line with no '=': ; Input line with no '=': ; Input line with no '=': ; Final record not terminated by '='
The = I placed at the end doesn't have spaces and it is followed by a newline.
and it is followed by a newline.
Remove this new line.
Perfect, thank you. Now it went smoothly.
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