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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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Primer3 issue with the Sequence_ID #51

Closed Kisekya closed 2 years ago

Kisekya commented 2 years ago

Hi everyone,

I am trying to sign primers for my sequences but I have an error that I do not understand the problem.

I made my finputfile.txt and I made the command below:

aka@aka:~/Desk/Primer$ /home/aka/primer3/src/primer3_core < input.txt > result.txt

input.txt

PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=23
PRIMER_MAX_NS_ACCEPTED=1
PRIMER_PRODUCT_SIZE_RANGE=100-150
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/home/coralie/primer3/src/primer3_config/
=
SEQUENCE_ID=Qrob_P0702440.2
SEQUENCE_TEMPLATE=ATGCCTCCATCCTTCATCCACCGCCTACTCATCTCT....AATTGAGGAAGAGGGATTGTTCAATGAGTGGGAAGATTGCAAGACTTTGTTGA
=

It comes out: a results.txt file below, I feel like it's working but I have an error that shows up.

result.txt

    PRIMER_TASK=generic
    PRIMER_PICK_LEFT_PRIMER=1
    PRIMER_PICK_INTERNAL_OLIGO=0
    PRIMER_PICK_RIGHT_PRIMER=1
    PRIMER_OPT_SIZE=20
    PRIMER_MIN_SIZE=18
    PRIMER_MAX_SIZE=23
    PRIMER_MAX_NS_ACCEPTED=1
    PRIMER_PRODUCT_SIZE_RANGE=100-150
    P3_FILE_FLAG=1
    PRIMER_EXPLAIN_FLAG=1
    PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/home/coralie/primer3/src/primer3_config/
    PRIMER_ERROR=Need PRIMER_SEQUENCE_ID if PRIMER_FILE_FLAG is not 0
    =

When I change the SEQUENCE_ID to PRIMER_SEQUENCE_ID it doesn't work at all and shows me this:

PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=23
PRIMER_MAX_NS_ACCEPTED=1
PRIMER_PRODUCT_SIZE_RANGE=100-150
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/home/coralie/primer3/src/primer3_config/
PRIMER_ERROR=Need PRIMER_SEQUENCE_ID if PRIMER_FILE_FLAG is not 0
=
PRIMER_SEQUENCE_ID=Qrob_P0702440.2
SEQUENCE_TEMPLATE=ATGCCTCCATCCTTCATCCACCGCCTACTCATCTCT....AATTGAGGAAGAGGGATTGTTCAATGAGTGGGAAGATTGCAAGACTTTGTTGA
PRIMER_ERROR=Need PRIMER_SEQUENCE_ID if PRIMER_FILE_FLAG is not 0
=
PRIMER_SEQUENCE_ID=test2
SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
PRIMER_ERROR=Need PRIMER_SEQUENCE_ID if PRIMER_FILE_FLAG is not 0
=
PRIMER_SEQUENCE_ID=test3
SEQUENCE_TEMPLATE=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
PRIMER_ERROR=Need PRIMER_SEQUENCE_ID if PRIMER_FILE_FLAG is not 0
=

Do you know where this error comes from? Are my primers good in the first case with the error?

Thank you in advance for your help

Have a nice day

Kisekya

untergasser commented 2 years ago

Hi,

which version are you using and can you send the complete input and output?

Maybe there is a bug, I would like to track it down.

Best,

Andreas

Kisekya commented 2 years ago

Hi, Thank you for your help!

I use the version on the github.

input


PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=23
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_PRODUCT_SIZE_RANGE=100-150
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/home/coralie/primer3/src/primer3_config/
=
SEQUENCE_ID=Qrob_P0702440.2
SEQUENCE_TEMPLATE=ATGCCTCCATCCTTCATCCACCGCCTACTCATCTCTACTTCCACTCTCCGCAACACCACCCACCTCTCCTCTCTCCACCTCAACCTCACCCTCCTCTCACTCCACCACCACCGCCACCGCCACCGCCACCGCTCTCTCTCCTCCACTCCCACTCCACCACCGCCACCACCACCATCACCCTTCCACCCCAGAAGCAAAACCCAACTCGAAACCCAGTTCGAAACATGGGTCCAAAACCTCAAACCCGGGTTCACTCCGTCCGATGTCGAGCACACCCTTTGGTCCCAATCCGACCCGGACCTCGCTCTCGACCTCTTCCGCTGGACTACCTTACAACGTGGCTACACTCACACCCACGCCACCTACTTCACCATCATCAAAATCCTCGTCTCCAACAAGCGCTACGGCCTCGCCGAGACCCTCATCGAAGAGGTACTCTCTGGTGCTTGCGACATTAACATTCCTCTTTATAACTACATTATCAAGTTCTGTTGTGATAAAAGATCGCTCTTTAATCGAGCCTTTGATGTTTATAAGAAAATGTATAATTCCGAGAATTGTAAACCCAATTTACAGACTTATTCCATGTTGTTTAACCTGTTGCTTAGAAGGTTCAATAAGTTGAATGTTTGTTATATGTATTTGCAATCGGCTAAGTCGTTGTCGAAGCAAATGAAGGCCGCGGGTGTGATTCCGGATACTTATGTGTTGAATATGATTATAAAGGCGTACTCGAAATGTCTTGAGGTTGATGAGGCCATTCGGGTTTTTCGTGAAATGGGGTTGTATGGATGTGAGCCGAATGCATATACTTACGGGTATATGGTGAAGGGGTTGTGTGAGAAAGGGAGGGTTGGTCAGGGTTTTGGGTTTTATGAGGAAATGAAGGGGAAAGGTTTGGTTCCAAGTAGTAGTAGTTATATGATTTTGATTTGTAGTCTTGCGTTAGAACGAAGGTTTGAGGATGCTATTGGGGTTGTTTTCGATATGTTGGGGAATTTTATGGGTCCTGATTTGTTGACTTATAAAACGTTGTTGGAAGGTTTGTGTCGTGAAGGGAGGGGCAATGAGGCCTTTGAATTGCTTGATGAATTGAGGAAGAGGGATCGTTCGATGAGTGAGAAGATTCTTGCATTGGAATGGTGGTTTGAGGATGTGATTGAGGTTGTGTTTGATATGTTGGGGAATTCGATGGGTCCTGATTTTTTGACTTACAAAACCTTGTTGTGTCGGGAAGGGAGGGGCGGTGAGACCTTTGAACTTCTTGATGAATTGAGGAAGAGGGATTGTTCAATGAGTGGGAAGATTGCAAGACTTTGTTGA
=
SEQUENCE_ID=Qrob_P0255410.2
SEQUENCE_TEMPLATE=ATGTTAAAGGTCCCGTGCAATATGTTGAATGACCAAATCCAAGTGATAACGCAATGTGGGTTTATGGGCCGCGGGTCCATTTTGACATGTGGGCACCAGAGAAATGCCGTCGTCTTAGGCTCTAGTTCTGGTTTTGGTTCTTTGAACAAAGGTCCAATTATGGCTGTGGTGTCAAACCGGATTGTTGGATCCGGGACAGGGTCAGGATCAGGGACTCTGGCGGATCGGTTGACAGAAGATGGGTTGTCGTATAAGGAGAGATTTGTAGTGAGATGCTATGAAGTTGGGATTAACAAAACTGCCACTATTGAAACCGTTGCTAATCTCTTGCAGGAGGTTGGATGCAACCATGCACAGAGTTTAGGATTTTCAACAGATGGCTTTGCAACCACCCCCACCATGAGGAAATTACATCTCATATGGGTTATTGCCCGCATGCACATGGAAATCTACAAATACCCAGCTTGGAATGATGTGGTTGAAATTGAAACTTGGTTCCAAGGGGAGGGAAGAATTGGAGCCAGACGAGATTGGATACTGAAGGACTATGCTACTGATCAAGTTATTGGAAGAGCAACAAGCAAGTGGGTGATGATGAACCAAGACACCAGACGACTTCAGAAAGTTAGCGATGATGTACGGGATGATTATTTAGTTTATTGTCCCCGAGAACCCAGATTAGCAATTCCTGAGGAGAACAGCAATAGTTTGAGAAGAATACCAAAACTGGAAGATCCTGCCGAGAATTTCAAGCGGGGACTTGTGCCCAGAAGAGCTGATCTGGACATGAACCAACATGTTAATAATGTCACTAACATTGGATGGGTTATGGAGAGCTTGCCTCAAGAAGTCATTGATAGCCATGAGCTGCAAGCTATTACTTTAGATTATAGACGGGAGTGCCATCACAATGACAGAGTTGATTCACTTACCAGTGTGGATGCCAAAGCAGTTTCTAAGATTGAAGGAACTAATTATGGGTCTACAGCTGCAACAGAAAACAAAGAAGATTTACGTCAGTTTCTACATTTATTGAGAGTATCAGGTGATGGGCTTGAAATCAATCGAGGACGCACTGAGTGGAGAAAGAAACATGCAAGATGA
=

Output


PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=23
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_PRODUCT_SIZE_RANGE=100-150
P3_FILE_FLAG=1
PRIMER_EXPLAIN_FLAG=1
PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/home/coralie/primer3/src/primer3_config/
PRIMER_ERROR=Need PRIMER_SEQUENCE_ID if PRIMER_FILE_FLAG is not 0
=
SEQUENCE_ID=Qrob_P0702440.2
SEQUENCE_TEMPLATE=ATGCCTCCATCCTTCATCCACCGCCTACTCATCTCTACTTCCACTCTCCGCAACACCACCCACCTCTCCTCTCTCCACCTCAACCTCACCCTCCTCTCACTCCACCACCACCGCCACCGCCACCGCCACCGCTCTCTCTCCTCCACTCCCACTCCACCACCGCCACCACCACCATCACCCTTCCACCCCAGAAGCAAAACCCAACTCGAAACCCAGTTCGAAACATGGGTCCAAAACCTCAAACCCGGGTTCACTCCGTCCGATGTCGAGCACACCCTTTGGTCCCAATCCGACCCGGACCTCGCTCTCGACCTCTTCCGCTGGACTACCTTACAACGTGGCTACACTCACACCCACGCCACCTACTTCACCATCATCAAAATCCTCGTCTCCAACAAGCGCTACGGCCTCGCCGAGACCCTCATCGAAGAGGTACTCTCTGGTGCTTGCGACATTAACATTCCTCTTTATAACTACATTATCAAGTTCTGTTGTGATAAAAGATCGCTCTTTAATCGAGCCTTTGATGTTTATAAGAAAATGTATAATTCCGAGAATTGTAAACCCAATTTACAGACTTATTCCATGTTGTTTAACCTGTTGCTTAGAAGGTTCAATAAGTTGAATGTTTGTTATATGTATTTGCAATCGGCTAAGTCGTTGTCGAAGCAAATGAAGGCCGCGGGTGTGATTCCGGATACTTATGTGTTGAATATGATTATAAAGGCGTACTCGAAATGTCTTGAGGTTGATGAGGCCATTCGGGTTTTTCGTGAAATGGGGTTGTATGGATGTGAGCCGAATGCATATACTTACGGGTATATGGTGAAGGGGTTGTGTGAGAAAGGGAGGGTTGGTCAGGGTTTTGGGTTTTATGAGGAAATGAAGGGGAAAGGTTTGGTTCCAAGTAGTAGTAGTTATATGATTTTGATTTGTAGTCTTGCGTTAGAACGAAGGTTTGAGGATGCTATTGGGGTTGTTTTCGATATGTTGGGGAATTTTATGGGTCCTGATTTGTTGACTTATAAAACGTTGTTGGAAGGTTTGTGTCGTGAAGGGAGGGGCAATGAGGCCTTTGAATTGCTTGATGAATTGAGGAAGAGGGATCGTTCGATGAGTGAGAAGATTCTTGCATTGGAATGGTGGTTTGAGGATGTGATTGAGGTTGTGTTTGATATGTTGGGGAATTCGATGGGTCCTGATTTTTTGACTTACAAAACCTTGTTGTGTCGGGAAGGGAGGGGCGGTGAGACCTTTGAACTTCTTGATGAATTGAGGAAGAGGGATTGTTCAATGAGTGGGAAGATTGCAAGACTTTGTTGA
PRIMER_LEFT_EXPLAIN=considered 7270, GC content failed 170, low tm 3556, high tm 1208, high hairpin stability 267, long poly-x seq 18, ok 2051
PRIMER_RIGHT_EXPLAIN=considered 7270, GC content failed 170, low tm 3738, high tm 1189, high hairpin stability 337, long poly-x seq 18, ok 1818
PRIMER_PAIR_EXPLAIN=considered 163, unacceptable product size 156, ok 7
PRIMER_LEFT_NUM_RETURNED=5
PRIMER_RIGHT_NUM_RETURNED=5
PRIMER_INTERNAL_NUM_RETURNED=0
PRIMER_PAIR_NUM_RETURNED=5
PRIMER_PAIR_0_PENALTY=0.071705
PRIMER_LEFT_0_PENALTY=0.036388
PRIMER_RIGHT_0_PENALTY=0.035317
PRIMER_LEFT_0_SEQUENCE=CACGCCACCTACTTCACCAT
PRIMER_RIGHT_0_SEQUENCE=AATGTCGCAAGCACCAGAGA
PRIMER_LEFT_0=354,20
PRIMER_RIGHT_0=455,20
PRIMER_LEFT_0_TM=60.036
PRIMER_RIGHT_0_TM=59.965
PRIMER_LEFT_0_GC_PERCENT=55.000
PRIMER_RIGHT_0_GC_PERCENT=50.000
PRIMER_LEFT_0_SELF_ANY_TH=0.00
PRIMER_RIGHT_0_SELF_ANY_TH=0.00
PRIMER_LEFT_0_SELF_END_TH=0.00
PRIMER_RIGHT_0_SELF_END_TH=0.00
PRIMER_LEFT_0_HAIRPIN_TH=0.00
PRIMER_RIGHT_0_HAIRPIN_TH=37.18
PRIMER_LEFT_0_END_STABILITY=3.5500
PRIMER_RIGHT_0_END_STABILITY=3.1000
PRIMER_PAIR_0_COMPL_ANY_TH=0.00
PRIMER_PAIR_0_COMPL_END_TH=0.00
PRIMER_PAIR_0_PRODUCT_SIZE=102
PRIMER_PAIR_0_PRODUCT_TM=85.1
PRIMER_PAIR_1_PENALTY=0.071967
PRIMER_LEFT_1_PENALTY=0.040351
PRIMER_RIGHT_1_PENALTY=0.031616
PRIMER_LEFT_1_SEQUENCE=CTCACCCTCCTCTCACTCCA
PRIMER_RIGHT_1_SEQUENCE=TTGGGTTTTGCTTCTGGGGT
PRIMER_LEFT_1=84,20
PRIMER_RIGHT_1=203,20
PRIMER_LEFT_1_TM=59.960
PRIMER_RIGHT_1_TM=60.032
PRIMER_LEFT_1_GC_PERCENT=60.000
PRIMER_RIGHT_1_GC_PERCENT=50.000
PRIMER_LEFT_1_SELF_ANY_TH=0.00
PRIMER_RIGHT_1_SELF_ANY_TH=0.00
PRIMER_LEFT_1_SELF_END_TH=0.00
PRIMER_RIGHT_1_SELF_END_TH=0.00
PRIMER_LEFT_1_HAIRPIN_TH=0.00
PRIMER_RIGHT_1_HAIRPIN_TH=0.00
PRIMER_LEFT_1_END_STABILITY=3.8600
PRIMER_RIGHT_1_END_STABILITY=4.9500
PRIMER_PAIR_1_COMPL_ANY_TH=0.00
PRIMER_PAIR_1_COMPL_END_TH=0.00
PRIMER_PAIR_1_PRODUCT_SIZE=120
PRIMER_PAIR_1_PRODUCT_TM=90.1
PRIMER_PAIR_2_PENALTY=0.072114
PRIMER_LEFT_2_PENALTY=0.040499
PRIMER_RIGHT_2_PENALTY=0.031616
PRIMER_LEFT_2_SEQUENCE=ACCTCACCCTCCTCTCACTC
PRIMER_RIGHT_2_SEQUENCE=TTGGGTTTTGCTTCTGGGGT
PRIMER_LEFT_2=82,20
PRIMER_RIGHT_2=203,20
PRIMER_LEFT_2_TM=59.960
PRIMER_RIGHT_2_TM=60.032
PRIMER_LEFT_2_GC_PERCENT=60.000
PRIMER_RIGHT_2_GC_PERCENT=50.000
PRIMER_LEFT_2_SELF_ANY_TH=0.00
PRIMER_RIGHT_2_SELF_ANY_TH=0.00
PRIMER_LEFT_2_SELF_END_TH=0.00
PRIMER_RIGHT_2_SELF_END_TH=0.00
PRIMER_LEFT_2_HAIRPIN_TH=0.00
PRIMER_RIGHT_2_HAIRPIN_TH=0.00
PRIMER_LEFT_2_END_STABILITY=3.5100
PRIMER_RIGHT_2_END_STABILITY=4.9500
PRIMER_PAIR_2_COMPL_ANY_TH=0.00
PRIMER_PAIR_2_COMPL_END_TH=0.00
PRIMER_PAIR_2_PRODUCT_SIZE=122
PRIMER_PAIR_2_PRODUCT_TM=90.1
PRIMER_PAIR_3_PENALTY=0.145327
PRIMER_LEFT_3_PENALTY=0.107017
PRIMER_RIGHT_3_PENALTY=0.038310
PRIMER_LEFT_3_SEQUENCE=AGGTTGATGAGGCCATTCGG
PRIMER_RIGHT_3_SEQUENCE=CCTGACCAACCCTCCCTTTC
PRIMER_LEFT_3=745,20
PRIMER_RIGHT_3=861,20
PRIMER_LEFT_3_TM=60.107
PRIMER_RIGHT_3_TM=59.962
PRIMER_LEFT_3_GC_PERCENT=55.000
PRIMER_RIGHT_3_GC_PERCENT=60.000
PRIMER_LEFT_3_SELF_ANY_TH=5.01
PRIMER_RIGHT_3_SELF_ANY_TH=0.00
PRIMER_LEFT_3_SELF_END_TH=0.00
PRIMER_RIGHT_3_SELF_END_TH=0.00
PRIMER_LEFT_3_HAIRPIN_TH=32.09
PRIMER_RIGHT_3_HAIRPIN_TH=0.00
PRIMER_LEFT_3_END_STABILITY=4.3000
PRIMER_RIGHT_3_END_STABILITY=2.6200
PRIMER_PAIR_3_COMPL_ANY_TH=0.96
PRIMER_PAIR_3_COMPL_END_TH=1.97
PRIMER_PAIR_3_PRODUCT_SIZE=117
PRIMER_PAIR_3_PRODUCT_TM=83.3
PRIMER_PAIR_4_PENALTY=0.147807
PRIMER_LEFT_4_PENALTY=0.107456
PRIMER_RIGHT_4_PENALTY=0.040351
PRIMER_LEFT_4_SEQUENCE=CCTCCATCCTTCATCCACCG
PRIMER_RIGHT_4_SEQUENCE=TGGAGTGAGAGGAGGGTGAG
PRIMER_LEFT_4=3,20
PRIMER_RIGHT_4=103,20
PRIMER_LEFT_4_TM=59.893
PRIMER_RIGHT_4_TM=59.960
PRIMER_LEFT_4_GC_PERCENT=60.000
PRIMER_RIGHT_4_GC_PERCENT=60.000
PRIMER_LEFT_4_SELF_ANY_TH=0.00
PRIMER_RIGHT_4_SELF_ANY_TH=0.00
PRIMER_LEFT_4_SELF_END_TH=0.00
PRIMER_RIGHT_4_SELF_END_TH=0.00
PRIMER_LEFT_4_HAIRPIN_TH=0.00
PRIMER_RIGHT_4_HAIRPIN_TH=0.00
PRIMER_LEFT_4_END_STABILITY=4.9400
PRIMER_RIGHT_4_END_STABILITY=3.5100
PRIMER_PAIR_4_COMPL_ANY_TH=16.00
PRIMER_PAIR_4_COMPL_END_TH=14.28
PRIMER_PAIR_4_PRODUCT_SIZE=101
PRIMER_PAIR_4_PRODUCT_TM=86.1
=
SEQUENCE_ID=Qrob_P0255410.2
SEQUENCE_TEMPLATE=ATGTTAAAGGTCCCGTGCAATATGTTGAATGACCAAATCCAAGTGATAACGCAATGTGGGTTTATGGGCCGCGGGTCCATTTTGACATGTGGGCACCAGAGAAATGCCGTCGTCTTAGGCTCTAGTTCTGGTTTTGGTTCTTTGAACAAAGGTCCAATTATGGCTGTGGTGTCAAACCGGATTGTTGGATCCGGGACAGGGTCAGGATCAGGGACTCTGGCGGATCGGTTGACAGAAGATGGGTTGTCGTATAAGGAGAGATTTGTAGTGAGATGCTATGAAGTTGGGATTAACAAAACTGCCACTATTGAAACCGTTGCTAATCTCTTGCAGGAGGTTGGATGCAACCATGCACAGAGTTTAGGATTTTCAACAGATGGCTTTGCAACCACCCCCACCATGAGGAAATTACATCTCATATGGGTTATTGCCCGCATGCACATGGAAATCTACAAATACCCAGCTTGGAATGATGTGGTTGAAATTGAAACTTGGTTCCAAGGGGAGGGAAGAATTGGAGCCAGACGAGATTGGATACTGAAGGACTATGCTACTGATCAAGTTATTGGAAGAGCAACAAGCAAGTGGGTGATGATGAACCAAGACACCAGACGACTTCAGAAAGTTAGCGATGATGTACGGGATGATTATTTAGTTTATTGTCCCCGAGAACCCAGATTAGCAATTCCTGAGGAGAACAGCAATAGTTTGAGAAGAATACCAAAACTGGAAGATCCTGCCGAGAATTTCAAGCGGGGACTTGTGCCCAGAAGAGCTGATCTGGACATGAACCAACATGTTAATAATGTCACTAACATTGGATGGGTTATGGAGAGCTTGCCTCAAGAAGTCATTGATAGCCATGAGCTGCAAGCTATTACTTTAGATTATAGACGGGAGTGCCATCACAATGACAGAGTTGATTCACTTACCAGTGTGGATGCCAAAGCAGTTTCTAAGATTGAAGGAACTAATTATGGGTCTACAGCTGCAACAGAAAACAAAGAAGATTTACGTCAGTTTCTACATTTATTGAGAGTATCAGGTGATGGGCTTGAAATCAATCGAGGACGCACTGAGTGGAGAAAGAAACATGCAAGATGA
PRIMER_LEFT_EXPLAIN=considered 6030, GC content failed 16, low tm 3511, high tm 497, high hairpin stability 549, ok 1457
PRIMER_RIGHT_EXPLAIN=considered 6030, GC content failed 16, low tm 3580, high tm 416, high hairpin stability 364, ok 1654
PRIMER_PAIR_EXPLAIN=considered 186, unacceptable product size 178, ok 8
PRIMER_LEFT_NUM_RETURNED=5
PRIMER_RIGHT_NUM_RETURNED=5
PRIMER_INTERNAL_NUM_RETURNED=0
PRIMER_PAIR_NUM_RETURNED=5
PRIMER_PAIR_0_PENALTY=0.068821
PRIMER_LEFT_0_PENALTY=0.035317
PRIMER_RIGHT_0_PENALTY=0.033504
PRIMER_LEFT_0_SEQUENCE=TGGGTCTACAGCTGCAACAG
PRIMER_RIGHT_0_SEQUENCE=TTTCTCCACTCAGTGCGTCC
PRIMER_LEFT_0=977,20
PRIMER_RIGHT_0=1087,20
PRIMER_LEFT_0_TM=59.965
PRIMER_RIGHT_0_TM=59.966
PRIMER_LEFT_0_GC_PERCENT=55.000
PRIMER_RIGHT_0_GC_PERCENT=55.000
PRIMER_LEFT_0_SELF_ANY_TH=15.27
PRIMER_RIGHT_0_SELF_ANY_TH=0.00
PRIMER_LEFT_0_SELF_END_TH=6.26
PRIMER_RIGHT_0_SELF_END_TH=0.00
PRIMER_LEFT_0_HAIRPIN_TH=34.12
PRIMER_RIGHT_0_HAIRPIN_TH=31.34
PRIMER_LEFT_0_END_STABILITY=3.1600
PRIMER_RIGHT_0_END_STABILITY=4.7900
PRIMER_PAIR_0_COMPL_ANY_TH=0.00
PRIMER_PAIR_0_COMPL_END_TH=0.00
PRIMER_PAIR_0_PRODUCT_SIZE=111
PRIMER_PAIR_0_PRODUCT_TM=80.0
PRIMER_PAIR_1_PENALTY=0.140230
PRIMER_LEFT_1_PENALTY=0.108615
PRIMER_RIGHT_1_PENALTY=0.031616
PRIMER_LEFT_1_SEQUENCE=CCAGAGAAATGCCGTCGTCT
PRIMER_RIGHT_1_SEQUENCE=GAGTCCCTGATCCTGACCCT
PRIMER_LEFT_1=95,20
PRIMER_RIGHT_1=216,20
PRIMER_LEFT_1_TM=60.109
PRIMER_RIGHT_1_TM=60.032
PRIMER_LEFT_1_GC_PERCENT=55.000
PRIMER_RIGHT_1_GC_PERCENT=60.000
PRIMER_LEFT_1_SELF_ANY_TH=0.00
PRIMER_RIGHT_1_SELF_ANY_TH=0.00
PRIMER_LEFT_1_SELF_END_TH=0.00
PRIMER_RIGHT_1_SELF_END_TH=0.00
PRIMER_LEFT_1_HAIRPIN_TH=0.00
PRIMER_RIGHT_1_HAIRPIN_TH=0.00
PRIMER_LEFT_1_END_STABILITY=4.1800
PRIMER_RIGHT_1_END_STABILITY=4.3400
PRIMER_PAIR_1_COMPL_ANY_TH=0.00
PRIMER_PAIR_1_COMPL_END_TH=0.00
PRIMER_PAIR_1_PRODUCT_SIZE=122
PRIMER_PAIR_1_PRODUCT_TM=84.0
PRIMER_PAIR_2_PENALTY=0.140230
PRIMER_LEFT_2_PENALTY=0.108615
PRIMER_RIGHT_2_PENALTY=0.031616
PRIMER_LEFT_2_SEQUENCE=CCAGAGAAATGCCGTCGTCT
PRIMER_RIGHT_2_SEQUENCE=AGAGTCCCTGATCCTGACCC
PRIMER_LEFT_2=95,20
PRIMER_RIGHT_2=217,20
PRIMER_LEFT_2_TM=60.109
PRIMER_RIGHT_2_TM=60.032
PRIMER_LEFT_2_GC_PERCENT=55.000
PRIMER_RIGHT_2_GC_PERCENT=60.000
PRIMER_LEFT_2_SELF_ANY_TH=0.00
PRIMER_RIGHT_2_SELF_ANY_TH=0.00
PRIMER_LEFT_2_SELF_END_TH=0.00
PRIMER_RIGHT_2_SELF_END_TH=0.00
PRIMER_LEFT_2_HAIRPIN_TH=0.00
PRIMER_RIGHT_2_HAIRPIN_TH=0.00
PRIMER_LEFT_2_END_STABILITY=4.1800
PRIMER_RIGHT_2_END_STABILITY=4.4600
PRIMER_PAIR_2_COMPL_ANY_TH=0.00
PRIMER_PAIR_2_COMPL_END_TH=0.00
PRIMER_PAIR_2_PRODUCT_SIZE=123
PRIMER_PAIR_2_PRODUCT_TM=83.9
PRIMER_PAIR_3_PENALTY=0.142775
PRIMER_LEFT_3_PENALTY=0.106894
PRIMER_RIGHT_3_PENALTY=0.035881
PRIMER_LEFT_3_SEQUENCE=CCCCGAGAACCCAGATTAGC
PRIMER_RIGHT_3_SEQUENCE=TCAGCTCTTCTGGGCACAAG
PRIMER_LEFT_3=663,20
PRIMER_RIGHT_3=778,20
PRIMER_LEFT_3_TM=59.893
PRIMER_RIGHT_3_TM=59.964
PRIMER_LEFT_3_GC_PERCENT=60.000
PRIMER_RIGHT_3_GC_PERCENT=55.000
PRIMER_LEFT_3_SELF_ANY_TH=0.00
PRIMER_RIGHT_3_SELF_ANY_TH=0.00
PRIMER_LEFT_3_SELF_END_TH=0.00
PRIMER_RIGHT_3_SELF_END_TH=0.00
PRIMER_LEFT_3_HAIRPIN_TH=0.00
PRIMER_RIGHT_3_HAIRPIN_TH=43.06
PRIMER_LEFT_3_END_STABILITY=3.0900
PRIMER_RIGHT_3_END_STABILITY=3.1600
PRIMER_PAIR_3_COMPL_ANY_TH=18.98
PRIMER_PAIR_3_COMPL_END_TH=18.98
PRIMER_PAIR_3_PRODUCT_SIZE=116
PRIMER_PAIR_3_PRODUCT_TM=83.1
PRIMER_PAIR_4_PENALTY=0.142917
PRIMER_LEFT_4_PENALTY=0.106894
PRIMER_RIGHT_4_PENALTY=0.036022
PRIMER_LEFT_4_SEQUENCE=CCCCGAGAACCCAGATTAGC
PRIMER_RIGHT_4_SEQUENCE=AGCTCTTCTGGGCACAAGTC
PRIMER_LEFT_4=663,20
PRIMER_RIGHT_4=776,20
PRIMER_LEFT_4_TM=59.893
PRIMER_RIGHT_4_TM=59.964
PRIMER_LEFT_4_GC_PERCENT=60.000
PRIMER_RIGHT_4_GC_PERCENT=55.000
PRIMER_LEFT_4_SELF_ANY_TH=0.00
PRIMER_RIGHT_4_SELF_ANY_TH=0.00
PRIMER_LEFT_4_SELF_END_TH=0.00
PRIMER_RIGHT_4_SELF_END_TH=0.00
PRIMER_LEFT_4_HAIRPIN_TH=0.00
PRIMER_RIGHT_4_HAIRPIN_TH=0.00
PRIMER_LEFT_4_END_STABILITY=3.0900
PRIMER_RIGHT_4_END_STABILITY=3.0100
PRIMER_PAIR_4_COMPL_ANY_TH=18.98
PRIMER_PAIR_4_COMPL_END_TH=18.98
PRIMER_PAIR_4_PRODUCT_SIZE=114
PRIMER_PAIR_4_PRODUCT_TM=83.0
=

It generates also two others files for each samples with it: -Qrob_P0255410.2.for -Qrob_P0255410.2.rev -Qrob_P0702440.2.for -Qrob_P0702440.2.rev

Best

Kisekya

untergasser commented 2 years ago

Hi,

thank you for bringing this to our attention. Actually the error message was still using the old tags. It is fixed in: https://github.com/primer3-org/primer3/commit/dd9c69cfbcb9c68e614dc1b896afb580cd4d885f

Your file should also not have the first = And the config folder is optional if you do not need your own parameters. With the right tags, the primers are fine.

crash.txt

Best,

Andreas