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primer3-org / primer3

Primer3 is a command line tool to select primers for polymerase chain reaction (PCR).
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SEQUENCE_PRIMER_PAIR_OK_REGION_LIST doesn't honor specified values #81

Open kubu4 opened 1 month ago

kubu4 commented 1 month ago

I have the following parameters file:

SEQUENCE_ID=LOC120027825
SEQUENCE_TEMPLATE=TttttCTCtCCAttACAAttttAtCCCTtAAAAtATtTtAAtTTACACCCCAtCTACCCAtTCACCtTCTTTCACTATCCtCAACCCTCCACTtttACAATCtTCAACAATACATTCAAtCCTTCATtTACTCCttttCCtCtAATAATTTCAtttACCAAtACTTCtCCAtCtAATTACAAATCCCCTtCtTtAAATtTCCCATCCCTCTCCATCTtCTCCttCCAtTTttttTATCAtATCAAtCCCATTCTACTtCAttTTttttTtAACCACTCAtACTCTTAtTTTTCTTTTtATCACTtCTCCCtAtAACCTtCTTATCCTAtttTACCCCTttCTAtTtCTACATAACCCACAATTTTTCTttTCCACtttACACATtCTAtACTttttTTtTTCAACATATtACATTAAAATtACAtATCtCTATAATAtTTtTACACTCATTAAAttCCCAtTtCAtTCAACtTTATtTtATCAtTtTTATTTCCTtATAtTTtCTttTTtAAAATACAATCTACACTttACCTTTTAATCAtCtttTTTtCATtttTtttAtTTTTttCTTTCCATtATtACATCACCATtCttTAAATTtATTAATAtACCAATAACAtAtTTCCAAACTTCTTTtCCAATAACAtCAATTTTTCAtTTTTTCCCTCCCCACTCAAACCACTCCCAtACAtTCCTTtCAAAATTCTTtTTTtTtAATtTTTTTTtTTtTTtCTAAAAAtCTATTCTTTCCCATTTTAATttAACTCTATTACAtTAATtTACTTAATTtTTACCCAtAAATtATTTtATATTtTTATAAAAACttCTtCAtTttACCTTTAATCTtCATCAAAATATCTtTTCAATAtAAATTtAtAAtATAAAATACACtCTCtCTtTCTTTATTtACATttTCAtCTtTttATtCTTCCTTTttCTtAttAATtACACCATtTTttCTTATtACCAttCATAtACCACTATCTAATCAACCTttACTCtttTAtACATAAAtATtAATCCtttACACTCCAATTAtTATtATATTTTACtTTTCtTATttTATtTATTAtTTTtTttATtTCCATCATCCATTTTtTATtATATtTTACAAATTtCAATTCATACAATATtTTAAAAATTtCTATTTtTACAATATtTTACtAATTTtTAAACTTATtAAATtTTATtAATTCTAATTTtTTtTtttTAACATTAtTTACtTttTTAAAtCTAACATTAtCCTttTttCTAATtTTAtCTATtTTAttttTTAtttTCAtttTTtAAttTTAtAtTTtATtTttATATtAAtCTAtttTTACttTTCAAATTAtAtTTtAtCTttAATtTtTACATAAAtCTAtttTTATttATTTTtATtAATTAtATCAtCCTttTCTCATAtACTAtACtTAAATCCttAtCAtATAtTtTtAtCTCAAAtTATTTttACAtTtACACATTTtTTtTTtTTTTAtCTCTtAtCTCCAtCACTTTttATTTtAAATtATACTATTAttTTAtTtCtCAtACTtACAtCTTTAATTTtAtttTATTTTCATCCATATCAttTtAACCtTTTAtAAAAAtTtCTtTCACCAAAAtTATTttAACAATTTTACTTATATtttTATTAAAtTAtTAAAAAtATAAtTATTTttACCCATATTCCTAtCACtCAATtACTACATCAAtCtTtTtACTCTACACATTTtTTttATtCATTTtCTtTTTtTTTTAtTTATtTTTCAtATTATTTTtTtCCCAATAtAAACAAATttTAAATAATtTATTtTtTAATTTTttAtTCACTTTTAtAtAAAtTTACAtACtCATAAATATCATAACCCCCCAAAAATtCTAAtCTCCCCTtTTATTtTAATttTtAtAttTTAtCATtTCTTttttTTATtATATTTtTtCATCTtTtACTTTCTCACTCATCATTATTCACTATTCATTCATTATTATCTtTAATCATttTAtCATCCACAATtTAtCAtTTTTTAtAAACCTATTCTTATTTACATTATTTTTTTAACTTAtCATtTTAtCTAACCCTAACCTTAATCCAACTCCTCCTAACTCCTTAACCTTCAAtTTTATTCAAAtTTTAtTCTTAtAAACACAtTtCATtAtAATTTAtCATtTtTTTCTtAAttAAAAACTTAAACtATtTCAAtCTCTCAtATAACACAAAACACTtttTtAtTTTTCCTTATTtCAttTCATTTttATCAtACTCTtTAAAACACTtTAttAAAACAACCCTtTTTtTTTTTTTttTCTCTTTTTATCttCATtAAATTtTtATAATATTtTAAttTTtttTTtTtCCtTttCttATATCTTTtTtttCTATACTCttCCTTtTCTtAttATttTAttTTttTttTTtAAtAAATCCCTCTAtTttTtTtttttCTtTtCTTTttCAAAtTtttTttttTTATATCCTTCCTtTTTttCCCTtTCCtttttTATCATCttATttttCCACAtTtTCTCCTtACCCCTCCTtTCTCAtTATTTATtCTtCAtTAtTTTATtTtTCttttttCTAtttTCAtTTTtTTATATCTttAtTACTTCTCCTtTCTTATCCttTtTCCTtTtTtAATTTAAtTATtCTCTCTCTAATTCTCTCTTTCTTTCTCTCTCTCttAttACCTtAtCCCTAttACCATtCCTCAttACTACCTttCACtATtACTCCTTtCTtTCCCCAtTCCACCTttCCtTtCTtCTtCTCCAtTTTCAACTtTTCTtCCTtTttCTATttAACCCTtACCTtTTCACCAtACtTtCTACCTtTCCCAtACCCtCTtTTTTCAACTCTCTAtAtACAtCAttAtCttTAtAtATACTCTTAATtATCttCTATtAAAAtCCAACTtACATTTACTTCTtAttTtCTtACTTtCTtCACCCTCtACAACTACTtTtATTATTATTATTTtACCATtCTttTCATTTTtAACATTTtAACATCTTttCCATtTTCTtTTATAATCTCCACCCttCACAtCCAAAAtAttACTttCCACCCCTCATAtCCTttTTCCTCTCTAttTTTCTTCCTAttTTTTttCCTTTCTAtttAtTTTTTCCTAtCCACCtTtCTTCTACACCTtCATTtCTTtCTtTTTttttTTTTAttCTtttTTTCTtTACAtCACTTTtAtATATCAtCTtATtTAAtAAtttCTATATAAATACATTTtATTTtATTTtATAAttTCACTAtTTACTTATTAAATATttATATtTCTtAACtACTAACCCTtAtACACATCAtAAAtCTtAtTTCACtTCATAATATTtTtATTATAATCACAATCCCTTCTTACACCACTATAAATtTtTCTCTtTCCTTATCTtAtTCTCTtAATtTAtCCTATtTtTtACCTtAATtTATCTtTTACCTTAATtTAATtTAtCCTACATtTtTTACTTtAAAAATAACCCTAAATTttTTCTTATTCCCAACACAtCTCTtttATtTtTAATttTCATATTTATTACCATACCTCTCCACTTCTTCCTCTTTCCCTTTACAATTTTTATTTTTTTTAAATAATAAAtTtATtATTTATTTATTttTTAAAtAttAAAATCCTCACATCACAtAATttTtCTtTCCTCACACTtttAAtAtCAttAAtCCACTtAAttTtTTtTttTTATCATttCTATCATttAtACCCTttTCTTCAtttAtACtAAttTAtACCACATCCTCCTTCTCCAtCTCTAtTtTCAACtCATTAtATATtTACTtCCAACCCCCAACTtTATTTCTTTCTACATTATATAtAACTCTTTtATTATTtTtAAACATCATtATtCCCATtAtTTtTtttTttCtttATtACATttCCtTtAATCTtAAtAAtTAtACTCCTTTCACTtATtCTtTtAAtATtCCTtCATCAtAttAtAAATAtAtTtATATCAtCACTtTCTtTCTCATtTTtCTtTAtCTttAATtTTTTTAACAAATAAACTCAtCAAAAAAAtAAATtTCCCTTTTCAttACCCTtTCTTTCAAAtATAATTCtTAAAAATCCAAATAACTTCACAtATCTTCATTTTAAAtttTTTAAACACTtTTTCCCATtCTTCTTCAATtAACCATAAACAATTAATtAACATtCACCTtTttAACttTCtTTAAtACACTAACAtCTTACAtACttTAttCAtTTAttCCAAAtTTATtAAAACTTAttACACTAAAtAttCCTTTCTACTtACACTtAAAAACACCAAAAtAAAtATtTCCAtttTCCCTtCTCATCTtCAttAACtTtCCTTAtACATtCTtCAAttAttCATtAttACTACAtATtTttCCAtttCAATAAATTtCAATtTCCtTACTtTtAtACACCTAACACAtCtCTACAtttAtACAttACttACAtCTtATCtTCCTCtCAtTtACAtACCACtTtTAACAACACCTtCACAttATCttTACATCCtAACATCACACCTtCtttACAttTACCttATttCAACAACAACTtCCCtAtTTACACCAttAACtCACAATCCCTCCATCAtTtCTCAtACTtTCTtCAATAttCTAAtAtAttTTttACTtAtttCTTtTAttCCTtTTtTCAttCAttTCATCACCttCAACTACtTCtCCTATtttCACAAACCCACCtTCACTttACCAtACAtCCtCttTTTTtTCTCACCAttttTtATttTCttAATTtCtTTTATCtTTtAAttAATtAtCtTTACACTtAttCCTtTACTCTttAtCtttATCtATTTttAttTttAtttTCCtTCATttTCTttttCttTtTATCACAtCATCATCttACTtAtCTTtTTtTCTTTtCAttAAATCTCAACtCTtTtCtTTACAtttAAtACATCCTCCTCCCTCATATttTACCCTTCCTtCAttCTCATCCTtACATtACCCTCCAtCATtACAATtCCACCAtCCATACTtCTCATtTTtTtCtTtATTTCCTtCAAtACAttAATtTCAtTtTTCTtCCATttCCAtCtAAtAtCCCAtATCTCAATCCCATTtAtCACtTCTtttACCTTTTttATCttAtttTtAtAtCTAtttCCATTCtCCACAtAAATtTCTtttAACTTtCAttTtCCTTttTttAAtAtTttttTAACATCTCACAtCAAtAACTttCAAATCTttTtCAtTCCATtAttAttAtATtCACTtCAtTACTTAATtCAtCTttTttCCACACCAtATACTtACTtTTACTTTTtATTTTtACCCCACCTTTtTTCAtttACACATTATTCCATTTCTtTTAtTCACATtTCTtTttAACTTtTTCAtTTTATtTCTCAtTTtTTtAATATTtTTATtTTCATACAAATATTTACACATtTTAAtTTtACTtAAAATAAACtCAtTTtACAtTtAtAttACtTTTCTTTTAACAtTtAtAttAtTTTAtTATAATTAAtTCAtttACTAttTAACACAAATTACCACCAAACACAATAAtTATCAAACTAAACATTtTTATAACTAAAATAAAATCAtttTtACAtATTAAtTAACATAAAATATtTCATTAAAAAAAATtACCtTAtACATACAtTACTAtTCAAAAtTTTttACATACTTAtAttTtTtttTTTTTCTTTATTTTTACTATTTTCTACATTtTAtCATAATAtTtAAtACATAAACTATtAAATAACAAATATttAATCATtTAtTAACCAAAATAtTATTTAAAAAATCTAAATATATTTCAtATTTTTCAAAtTAtCCACCCTTTtCCTCtATtACAtCTTTtCACTCTCTTttCATTCTCTCTTCACCTttAATtCTTTTCCAAtAtTtTtCAAAACTtTCATCAAttCAAAtttTtACTACTTTAAAtAATtTCAAATATAAAATATATTTTtATTTtTTTTATACTTTTTtttTTACTACATtATTCCATATtTtTTATTTAATAtTTTTtAAtTCTTCACTATTATTCTACAATtTAtAAAATAtTAAAATAAAtAATCCCTttAATAAtTAttTtTtTCCAAACTTTTtACTttTACTtTACtTtTTTttTATCATATAtCTAtTAtTTACAAAAtTAAACTCATTTAAAAtCTTtAAACtTAtAATTTtTCCCTCAtAATTATtTCCACTCTtAAAATCTAtTtTCTTATCATtATTTTCAAAtTAAAATATAAAACAAtTTAtTtAAtTAAAATTATATACAtAtATTACTtAtAAACATtCAtCAAAATCATCCCAtAAtAATTACATTAAATTtCATTCATAAAAAATTATtATtAtATtACTtACTCAATATTTttCATTtTTttATtAATtTTATtCAACATTCAAATttATTTCCCTCTTtTTAtTTAtTACCTtTAATTtttTTtTAttCCTTtCCtATtTTttTtATtACTCTACTtAAtATCAtTtTAtTAtCAtTACTtAATttACCTACtTTTCCAtAtTCAtTCAAACCAtCAtAtAAttCCACAtTTttTCTtCCTtTtAAAAtAAtTTtATTATTTAAAAttACAATCTtAtATTtAAATAACAACAAttCAAATtCTTACTTATCCACCTtTATCAATCCAATTTACAAATtCCTtATATTTCTtACATtACCTtCTTTCTCTTTCTCCAtCTCTTCCACCTTtCTCTTCTttAtCTtCAtTTCAtTCTTttTtACtCTCAtCTCCTtTCTCTtTTCCACCATCATtTCTCTCAtCTCCTCCAtCTTAtACCAtATtTCAttttTCTTtTtttCCtTCTCTtTAtCTTCAtCCCATtCCCCAAACAtAtAAAACAtCAtCAtAtCTACAtCACCTCTCATTCTtAAACtACACATCTTCTtAAATtATCTAtTCTTTtAttCTAtAAATTTACAttATTAAAtTAttAtCTATATCTtTtTtAAATATATTtTtTCTCTtTttAAAAAAtAtttTTCTTAAATATCAtttAtTATTATTTtATTAttATTACTttTAttATTCTTTATTTtTTTTTACTttCAATtAttTtAAttTTTCTTtCtTCATTCACCAtTTtTTtTTACTtTATTTCtTCATACTTAATTTTtATAtTCCCCTAtATAtttAtTtttCtTACACATCACAtACAAACTAAAATttTCCACACAtACAtTtTttTAAAtAAttAAttCCAAAAAtATCATCAAttACAACAACCACCCtAtCCACTtCCTtTTCACACTtCTATCATCCAtAAttTtAttTCAtTACAttTACATCAAtCTtttACCtAtAtATTtAtAAACAtCTTCTATCTCAAttCCATCAtACTtCTAAACAtCAATCATTAACTtAtAtAtTCTtCTtCCCACATTtAtAACCAATCACAttACACTTTAATAAATttATCACTAtTCACTTTAAACAATtCCACTTTAAATAATttCACTTTAATAATtCTTAAATATCTTACATTACTCATTTCACATtTATATACTtTATTTTATACCATCTACTtCACCTTtCCTATtCCtCTCtCATTAACATCTtCTAACCATtTtTATtTtACCAATAAtATTTtATTTtATATCTtTAAATtATCTACAtATtATCAAACTAAtAAtAAACTATtAACTtCAACTCTtTTtATATTCAACTtCTTtTTAtttTTATttCAAttTCtAATTttCCtTCTtCCAAATttACTttACTTTTTTTTTTAttTttTTtAtCCttTCtAAATTtAAAAAAATTATATTATATACATATATACAAAATCCATATTtCtATAACAATAAATCtAACAATAAtTTTATTACATTAATtTtCATCACAtTTTTTTTATTATCATTTAATCTATTACTATTAtTTTtTTTtTAtTTTtTTTtTTAtCAAACTTATTTAATATCAAACTTCACTTTTCTCTAtCATAttCTACTTATCtTAATtAACtACATAAtCAAAATtCTTtCAATAAtTtATCttAATttTTttTtTAtATAATTTTTtTTTTATCtTCCTAtCTtTAtTTCAAAtTCAAACtCtCCATCAtTtAtCCAtCCTATATTCCTACTAAAtTTtTtAtttAtAtACAtCTAAATtAtCttAtTtAATTATttATtATtAttACAAAtAACCTtATTCTtTtTtCAAAtACAtTTCAACTtCAtCCAAAttAACAtAAttTAATTCTtTCtAttTTtAAAACAtAtAtACATACTtACAtAtATCTCTAtTTttATtTCAAATtTTTTTtCATttACATTtCCATTtAtttCTTCCACCATTTTAAAtTAtTCAACTtttTttttATTTCTATttATTtAtAATtATTAtCCAATtATCAtAtAATTATCTTAATCTTCAATTTttATTtCTTATttCTtTACATttCTTTAAtCTTCATCACCACCATtAAtTttCCACAATAATTtAATtACAttACTCCAtCACTtCtttTttCAtTAAATCACCAATTTtTTATTTAACACtTTCTCATTTACAtCAACtACCTttttAATAtTTACAttttAtAttAtttttATtAATTAtCCAATTtTAAACTttttATtATTAttTtACCATtATttTATtAtttCCAtATTtttAATTTAtCCAtttCTAAACACCCCTACTCTTATAATAATAATAATAATAATAATAATAATAATAAATtCCATtTtACCACAtAtTCAttACACCCATTTAACATCCCATCCAAAAtACAtCAACCTACACAtttCAATtTCCCCAATCACTtCCTTttttAATTtttATATTTTTTAtAtCAtACtAAAtAtTtCCTCCTACTttCCCtTCCAACACCACTCCCAtCAtCATCTttTCTCCCATCCAtttCCCAACCTTtCTTAtCTTCAtAAtCAAtCCAtCAtTtttATttAtttTTtTATtCTTCTttCAAtCTAATtTTTAtTCCTtAACTTATTCAttCTTtCCATAACAAAttttTTtAATACTTTTTtACTCAAtACATTTCAtCTTTTCATTtTAAATACATTTtTAAAAATAAAAAAAATCCAACTAAATTCCACTTTtACATTtTAtttTAATATtTtTAtATCAtTtACACAACATCTACATTTAATCAACTATATATTCAttCTtTCACACAACAAAATtTttAAAAAtTCAAttttAtTtAATACTTTTTTTTATAtCTTTtTTATAAACATTATACTACATTATAATAACAACTTtCCAAAAAACTTtAAATttAAACAAATAtAATTTTACAACAAAATCAATtTTTCTtttACAAAATAttATTAtTtACAtCTtTAATTAAAttAAtATCTTCTttCTACAtTAACAATtACAtCTACATAAAAAAAAAAAAAAAAAtCTAtTCAttTAACTACtTAtTTAATtTTtCAATTtTTTACAtCTTTCAtATTTtCTATCATtAATTTtTTAAttCCAAtttTAAtAAttAAAATttCATtAATTttAACCTAATtACTAACTCCTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtTtCATACATtTTCATCTATCACTTTtTtTAtTTAtCTAtCAATTttCTACTAATtATTTtCCTtTCTACCCAtACTCCTtTCTACCCAtACtCCAAACtTTATtCCACtCCCACttACtTTAtTTTATtTAtTtAACtACttAtCAtCtAtTtTtAtTCtTCAttCTAATTAATAtTCCTACTtCAACTtTCTTtATTATtAtTAtATCTACAAAtTATtCCTTCCTttCAttATtATATAATTAATATTATTTtTATCAATTtttTCtttATTTtTTTAACAAACTCTtCCATCtTttTAtTCTtCAtAAACATTtCTAtCCAAACACAtTCCTCTCTCtCTAtttttTTTATTCAtACCCAAtTTATACCCACATAAATATAATTtTATTCTCTTTTTATtCACTTTTCTCTCTATtCtTATTCTtACCTTtAACTTACTCATttttACAAtCCAtTtTCAtCAAttTACCCTtttTtAttttTtATACtCCAtTtTATTCTtACCTTtACTTAATTCCCTACTAATTCTACAACCTTTATCAttCTAATATTCTtAATATCCTtTTCTCTCtATATAtTCCAtTtAtTTTtTCAACAAAATTCAAATtATAttTAAACCtTATATTCAAttATttATAAAtATAAATtTACTtAAAATCCTATTtAATAAAAACTCCACAAtAAAATCTtTTttCCAACAAtTtttAtttTTTTCAtCttTTtAATTAAATTTATTCAtAtCACtCCCAtCAtCAAAtCCCCAtCAtCAAAtACCtTTACACTACTtCATAAttTAAtTCTtAtTttTCCAAACACACCAAtAAAtTTtTt
PRIMER_TASK=generic
SEQUENCE_PRIMER_PAIR_OK_REGION_LIST=1,3500,6958,3300
SEQUENCE_TARGET=3500,3458
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=25
PRIMER_MAX_NS_ACCEPTED=0
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/home/shared/primer3-2.6.1/src/primer3_config
=

However, when I run Primer3, this is the header of the output:


No mispriming library specified
Using 0-based sequence positions
NO PRIMERS FOUND

SEQUENCE SIZE: 10458
INCLUDED REGION SIZE: 10458

TARGETS (start, len)*: 3500,3458
PAIR_OK_REGIONS (left_start, left_len, right_start, right_len)*: 6675,-3174,6958,-3174

Notice that the PAIR_OK_REGIONS listed do not match what is in my parameters file.

Interestingly, if I change SEQUENCE_PRIMER_PAIR_OK_REGION_LIST=1,3500,6958,3300 to:

SEQUENCE_PRIMER_PAIR_OK_REGION_LIST=0,3500,6958,3300

then the header of the output is this:

PRIMER PICKING RESULTS FOR LOC120027825

No mispriming library specified
Using 0-based sequence positions
NO PRIMERS FOUND

SEQUENCE SIZE: 10458
INCLUDED REGION SIZE: 10458

TARGETS (start, len)*: 3500,3458
PAIR_OK_REGIONS (left_start, left_len, right_start, right_len)*: 6675,-3175,6958,-3175