design PCR primers with primer3 on command line

Question

[marongiu.luigi]

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:

1. how shall I write the file to feed primer3_core?

2. Is there a way to avoid the file and give the data as arguments to primer3_core?

Thnak you

Answer 1

[finswimmer]

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

[marongiu.luigi]

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?

[marongiu.luigi]

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.

[finswimmer]

and it is followed by a newline.

Remove this new line.

[marongiu.luigi]

Perfect, thank you. Now it went smoothly.