Primer3 error:./primer3_core: Input line with no '=':

Question

[bioinfo89]

Hello!!

I want to design PCR primers for 300 contig sequences. For the same, I tried using primer3 standalone. following command I am using to run the tool:

./primer3_core -format_output -default_version=2 -p3_settings_file=settings_file -strict_tags -output=primer3_out contigs.fa

For the above command I get the error: ./primer3_core: Input line with no '=':

settings_file is as below:

Primer3 File - http://primer3.sourceforge.net P3_FILE_TYPE=settings

P3_FILE_ID=Default settings of primer3 version 1.1.4 PRIMER_TASK=generic PRIMER_PICK_LEFT_PRIMER=1 PRIMER_PICK_INTERNAL_OLIGO=0 PRIMER_PICK_RIGHT_PRIMER=1 PRIMER_NUM_RETURN=1 PRIMER_PRODUCT_SIZE_RANGE=100-600 PRIMER_MIN_SIZE=18 PRIMER_OPT_SIZE=22 PRIMER_MAX_SIZE=27 PRIMER_MIN_GC=45.0 PRIMER_OPT_GC_PERCENT=50.0 PRIMER_MAX_GC=58.0 PRIMER_MIN_TM=50.0 PRIMER_OPT_TM=55.0

PRIMER_MAX_TM=60.0

contigs.fa file is as below:

SEQUENCE_ID=c0_g1_i1_len=235_path=[1:0-234] [-1, 1, -2]

SEQUENCE_TEMPLATE=TTTTTGTCTGTCCTGCCCTGTCTGTCTCTCTGTCTCTTTCCAGCTGTGATTCTTCATCTTGTCCTCTCCCCGTCTCTCTCTCTTGCTCTTTATCTCTTTCTCTGCCTCTCTCTGTCATTCTCTCTCTCCCTGGCCCTCTCTGCCCAGATCG

SEQUENCE_ID=c0_g1_i1_len=220_path=[352:0-219] [-1, 352, -2]

SEQUENCE_TEMPLATE=AGATTAAAGCTTGCAAGAGCAGTTTGTAGATTATTTGAGGTAATAGTTTTTATGTTTTCAACAATGATGAAAAGTTTAATCGAAGGATATGGAGGAGAAAGGATCAACTCCACTCATCTGTGCCTACACATAGCTCTCTAAAGGTTGAGGGTAG

Any help for the same will be appreciated.

Thanks!

Answer 1

[Rajesh Detroja]

Hello,

I think you can run primer3 standalone programme as following:

1) Prepare input file

First of all you need to prepare input file for multiple sequences something like this:

for example save the following file as multi_seq_test

SEQUENCE_TARGET=37,21
PRIMER_TASK=pick_detection_primers
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=1
PRIMER_PRODUCT_SIZE_RANGE=75-100
P3_FILE_FLAG=1
SEQUENCE_INTERNAL_EXCLUDED_REGION=37,21
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/home/detrojar/src/primer3-2.3.7/src/primer3_config/
=
SEQUENCE_ID=test1
SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
=
SEQUENCE_ID=test2
SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
=
SEQUENCE_ID=test3
SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
=

Note: Please set path of your computer for "PRIMER_THERMODYNAMIC_PARAMETERS_PATH" parameter.

2) Run primer3_core programme

Now you need to run the following command:

primer3_core < multi_seq_test

This will generate output files for each sequences.

Note: Please set all the length parameters according to minimum and maximum length of your sequences. Follow the manual for more details.

Thanks, Rajesh

[bioinfo89]

Thanks Rajesh that was very helpful

Answer 2

[aboozar.soorni]

I think a problem could be the structure of ids. Remove equal sign and square brackets from ids and check how it works.

[bioinfo89]

I tried doing that also but It still shows the same error.

[aboozar.soorni]

Do you have an equal sign in the last line of setting and fasta files? Check example file!