primer3 parameter input

Question

[shira.zaltsman]

I have a few 50bp sequence of a bacteriaas genome, I am trying to find primers using primer3 on linux.

this is my input (in file "example.txt"):

SEQUENCE_ID=example
SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
SEQUENCE_TARGET=37,21
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=1
PRIMER_PRODUCT_SIZE_RANGE=75-100
P3_FILE_FLAG=1
SEQUENCE_INTERNAL_EXCLUDED_REGION=37,21
PRIMER_EXPLAIN_FLAG=1
=
SEQUENCE_ID=test1
SEQUENCE_TEMPLATE=GACTGATCGATGCTAGCTACGATCGATCGATGCATGCTAGCTAGCTAGCTGCTAGC
=
SEQUENCE_ID=test2
SEQUENCE_TEMPLATE=CATCATCATCATCGATGCTAGCATCNNACGTACGANCANATGCATCGATCGT
=
SEQUENCE_ID=test3
SEQUENCE_TEMPLATE=NACGTAGCTAGCATGCACNACTCGACNACGATGCACNACAGCTGCATCGATGC
=

i used this commend

sudo primer3_core -output /path/out.txt<~/example.txt

and this my output:

SEQUENCE_ID=example
SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
SEQUENCE_TARGET=37,21
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=1
PRIMER_PRODUCT_SIZE_RANGE=75-100
P3_FILE_FLAG=1
SEQUENCE_INTERNAL_EXCLUDED_REGION=37,21
PRIMER_EXPLAIN_FLAG=1
PRIMER_LEFT_EXPLAIN=considered 65, too many Ns 17, low tm 48, ok 0
PRIMER_RIGHT_EXPLAIN=considered 228, low tm 159, high tm 12, high hairpin stability 22, ok 35
PRIMER_INTERNAL_EXPLAIN=considered 0, ok 0
PRIMER_PAIR_EXPLAIN=considered 0, ok 0
PRIMER_LEFT_NUM_RETURNED=0
PRIMER_RIGHT_NUM_RETURNED=0
PRIMER_INTERNAL_NUM_RETURNED=0
PRIMER_PAIR_NUM_RETURNED=0
=
SEQUENCE_ID=test1
SEQUENCE_TEMPLATE=GACTGATCGATGCTAGCTACGATCGATCGATGCATGCTAGCTAGCTAGCTGCTAGC
PRIMER_ERROR=SEQUENCE_INCLUDED_REGION length < min PRIMER_PRODUCT_SIZE_RANGE
=
SEQUENCE_ID=test2
SEQUENCE_TEMPLATE=CATCATCATCATCGATGCTAGCATCNNACGTACGANCANATGCATCGATCGT
PRIMER_ERROR=SEQUENCE_INCLUDED_REGION length < min PRIMER_PRODUCT_SIZE_RANGE
=
SEQUENCE_ID=test3
SEQUENCE_TEMPLATE=NACGTAGCTAGCATGCACNACTCGACNACGATGCACNACAGCTGCATCGATGC
PRIMER_ERROR=SEQUENCE_INCLUDED_REGION length < min PRIMER_PRODUCT_SIZE_RANGE
=

from what I understand the primer3 didnt find primers in this input, how can I change the input parmeter so it will work?

[Pierre Lindenbaum]

sudo

there is no reason why you should use sudo

[shira.zaltsman]

please do not direct me to the manual. unless its specific subject there

[Santosh Anand]

sudo is used for running commands with higher (superuser) privileges. If you start running all the commands with sudo, there is a high chance that you may alter/delete some system files, creating troubles in loading and ruunig the OS. That's what Pierre intended by his comment.

Answer 1

[Pierre Lindenbaum]

your problem is clearly defined here:

PRIMER_ERROR=SEQUENCE_INCLUDED_REGION length < min PRIMER_PRODUCT_SIZE_RANGE

because target (where to pick the primer) length is 21

SEQUENCE_TARGET=37,21

while you want a PCR product of (min) 75

PRIMER_PRODUCT_SIZE_RANGE=75-100