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Primer3 is a command line tool to select primers for polymerase chain reaction (PCR).
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Final record not terminated by '=' value #16

Closed DeondeJager closed 5 years ago

DeondeJager commented 5 years ago

Hi guys,

I'm trying to run primer3 release 2.4.0 on Ubuntu 18.04 LTS using an input file generated by the program MSDB (Microsatellite Search and Building Database). But I get an error saying the final record is not terminated by an '=', but it is. See the output below (include the command I used).

deon@deon-ubuntu:~/Software/primer3/src$

./primer3_core /home/deon/Documents/Microsatellite_Design/Tri/primer_design_TRI_short.txt SEQUENCE_ID=1 SEQUENCE_TEMPLATE=gccgccgagctaggcctggatcggtcggtctcggcaatcgccgagataaagaggaactcgcgaactttcgttgaaacaatgggaggggcttggctgaggctattagcccggttttgatgccaggcgttatcaagagttgaagccgagccttcaacatcaaccttctttcaggccgccatcttgattccccttgctcccatCCGCCGCCGCCGCCGCCGCCGacgacgccgccgacgacgactaagttttgtaagcttcgaggaagttcgtagaaggagccggcaagcaagcgctattacgtcaactcattattccggtcattttcaataagcgatagtctttcgttggcaacaggcatactgcggccgctcatcaaccatattattaattgctaatattaaaagtcctttcacgaatcctt SEQUENCE_TARGET=201,21 PRIMER_MAX_END_STABILITY=250.0 PRIMER_PRODUCT_SIZE_RANGE=100-280

SEQUENCE_ID=2 SEQUENCE_TEMPLATE=aaatataattacttataatataattataaaaatacatttacttattaactatggaaattataaaattaatggaaattacttaatcaaatttattttcaatttttcaattttaatttataactttctaaacctaacaatagatacttttatattttatttactttcaccgcttttgtataaaaaaaaaacttactatgagcATAATAATAATAATAATAATAatggtattaacaataattacaaataataaaatttacagcagagtaatatgacattaatgttttgataataaagaccacattgttaaaaataaagaaaagctctccattgaatgaatacatatcaacgttattgtcgcatatgacaaaatagctttcatgctttcgagtaatatatttctatataatataattcaataagc SEQUENCE_TARGET=201,21 PRIMER_MAX_END_STABILITY=250.0 PRIMER_PRODUCT_SIZE_RANGE=100-280

; Final record not terminated by '='value: 50.0alue: 250.0

; Final record not terminated by '='value: 50.0Y value: `250.0

untergasser commented 5 years ago

Hi, this could be a newline windows vs linux issue. Could you send the input file so we could recreate the bug? Best, Andreas

untergasser commented 5 years ago

Hi, maybe this commit fixing windows newlines solved your bug report: https://github.com/primer3-org/primer3/commit/2faf322c025772d21dbf0cc7798a89c2c89919bd Best, Andreas

untergasser commented 5 years ago

Taking the input from above it run without errors in this version: https://github.com/primer3-org/primer3/commit/f382935091e1e49bcf2ce8afe8d4d034776509a1 So without the input file I cannot investigate further.

Best, Andreas

DeondeJager commented 5 years ago

Hi Andreas,

Thank you for your feedback! The commit seems to have fixed the issue, thank you!

I re-ran the above command with the same input file and with a different input file. I'm no longer getting the error and I'm getting the expected output.

Here is a link to the input file, if you would still like to test it on your side, but I think it has been solved and the issue can probably be marked as solved and closed! https://drive.google.com/open?id=1EQW_TLe160JsbLpIdHvcieHpcFQlXXJU

Best, Deon

untergasser commented 5 years ago

Hi Deon,

it was the windows '\r\n' newline (in your file) vs Linux '\n' newline (what old Primer3 expected) issue. Now Primer3 an handle both, so this issue is fixed. Thanks for reporting!

Best,

Andreas

326reborn commented 3 years ago

Hi Deon and Andreas,

I met the same problem as you in the Latest release. PRIMER_ERROR=Final record not terminated by '=' I'm sure my input file is the Linux '\n' newline by 'cat -A'. I got it by the misa.pl and p3_in.pl script which download from MISA. Looking forward to your reply.

Best,

Yu zhang test1 test1.zip

untergasser commented 2 years ago

Maybe your sequence is too big, try something up to 50000bp.

Best, Andreas

Adinivich commented 3 weeks ago

Hi all! I have the same 'primer3_core: Input line with no '=': ' error, with the folling kind of input file:

SEQUENCE_ID=NC_014426.2_774682-775882 SEQUENCE_TEMPLATE=GGCAGATGTCACTCTTCGATACCTTTACGTTCGACGGTAAGTAATATTCGACTCGCGCGGGCAAGGCGCGATGTAACAGATTGTTTCGTTGTTTCTATGAATTACATCCGGCTCCTCTGTGAGGAAAACCAAGCCATCATCGCCTCCTGGCGAACGGGTTAGGAATCCTCCCCGTCCGCGTATCCCTGAGCATCTCCCCCGATCTCGCGTGCTTATCcatcaatcgcgcgcgcaaaaagtccaccgccgccgtcggaggctccgccgtctcgtctccgcgcgcgccctccaACGCGACCACCTCGAATCCATCCCGCTCGTACGTCCGCCGCTTCGCCTCCCGTCGCTCCCTCTTACTCCTCTTCACCCGTTTCTTCCCTCCGACGTGcgcctcgtacgcgtcgaacgcccgtcttttctcgccgccgcgccgtccgtccccgagcgcgcccgcgacgacgtcctccgGAAGCGCCTCGagatcgtcttcgtccgccctcgcgtcgtccgccgacgccgtccacgccgtcgtcgtcgccgtttCCGTCCCATcgtcccctcgcgcgcgcgcgttcgaacgcgccgccggtcgtcgcgtcttcgccgccgccgccgccgctcgcaccgccgcgcgcgcgttcgcccgcgcccgcgtcgcctccgcgcgtccgcgcgccccgctCACTTCCTCGGGCGCGTCCGAGTCCGAGcgttcgtcctcgcccgcgagcgccgcgcgcgtcgagcgcggcgctcgcgNNNNNNNNNNNNNNNNNNNNGGGGGGGGCGGCGgggcggacgaggacgacggcccGGTGAgaccgcggtcgaggacgagcgcggagacgtcgcgggTGCGGTGTGATGGATGTCGGTGCACGCTGGCGTAtccggcgggcgcggcggcggtgcggtgcgcggtgtgcgggacggtgacgcggtgcgagacgggcgcgggacgtttggacggcggacgcgcggacgacgcgagcgccgctcgaggtggtcccgcgacgacggcgctgaCGATGGAGAACATGGTGGTGGTGGAGAATCCGCCGACGTTGACGGTGGACGGACGAGTGGTGACGAACATCGCGGTCGGGGTGAAACTCGACGATTGACTCGAcggtcgcggacgaacgacgcgacggtcgcgacgccgacgacatTTAGAGTCGGACGTATCGGATTCAATTTT SEQUENCE_TARGET=520,80 PRIMER_TASK=generic PRIMER_PICK_LEFT_PRIMER=1 PRIMER_PICK_RIGHT_PRIMER=1 PRIMER_OPT_SIZE=18 PRIMER_MIN_SIZE=15 PRIMER_MAX_SIZE=25 PRIMER_PRODUCT_SIZE_RANGE=250-600 PRIMER_GC_CLAMP=1 PRIMER_MIN_TM=58 PRIMER_OPT_TM=62 PRIMER_MAX_TM=66 PRIMER_OPT_GC_PERCENT=60

SEQUENCE_ID=NC_014427.2_79636-80836 SEQUENCE_TEMPLATE=TTCCGACACTGTCCGACCGCCGGTTTCGGTACTGAGCCGTGTATGAAAACAACCCCTAATATCTCAAGGGAAACAACGTGAAATACAAGTGAAATGGAAAAAAGTGTGAAAAACGTGAATTGTCTTTGCGCGCGTGTAGCTGTAAGAGGTCCGATCGAAGTTCGTGTGTTTTGGACGTGAATTTCAGCGAAAATAATTATTTGATGTGCAGTGTCAACACGTCATCATGGCACATCAGCGCGCATTTCTAGGGGCGGGGGACTTGCGCAAGACACCCCGAAACTGCCGCAATTTGCATTGCACTTGCATCTTGCAGGTGCAAAATGCCCCCATATTCCAGAGTAGGTAGGAAGATGCCATTTGTCCGAACATTATAATTATACTGTTTACCCATATGATCACCCTTAAACCCTAAACTAAACCCTAATCCACACGATAAtttgacgccgccgcccctGAATCGCGTGTCGGAGCCCCTTAAAAAcacccgcgtgtgcgggcgCCCACCCCTAATCTCTTGCGCAAGACGCCCCAAAACACCTGCAATTTGCACCTGCAATTTTGCTCTTGCATCTTGCAGGTAAAATTTCCCCCACCCCTAGGGACGTACAATCATCCAGACGAAACGAATCGCCCGTGGTGTCGCTGCGCAttggcgcggcgcgtcgattgCGTTGcgtgcgccgacgtcgacgcgcctcggtcgcggcggcgccgtcgcctcggtcgcacgcgcgtcgatcgatcgtcgaacgtcgatcgatcgcgtcgcgcgatggaggacgagcgcgaggattacggtgacgacgtcgacgacgtcgtcgacgcgctcgaggcggcggaggacgaggatgaACGAGACCTCGAGGGgggggacgcggacggacgGGTGAtcgtcgagagcgacgagcgaggcgacgcgctcggtacGCACGGTCGAAGAGGAACCGGTGGGGCGGTACCGAGACACttgcgaacgacgacgccgtacATGACGAAGTACGAACGGGCGAGGGTTCtgggaacgcgcgcgttgcagATCTCGATGAATGCGCCGGTGCTCGTGCagctcgagggcgagacGGATCCGTTGGAGATCGCGGGGAAGGAGCTCCGGGAGAAGAGAGTGCCGTTCGTCGTTCGAAGATATCTCCCGGATGGCTCGTACGAGGATTGGTGCATCGAGGAGCTGATCGTGCAC SEQUENCE_TARGET=520,80 PRIMER_TASK=generic PRIMER_PICK_LEFT_PRIMER=1 PRIMER_PICK_RIGHT_PRIMER=1 PRIMER_OPT_SIZE=18 PRIMER_MIN_SIZE=15 PRIMER_MAX_SIZE=25 PRIMER_PRODUCT_SIZE_RANGE=250-600 PRIMER_GC_CLAMP=1 PRIMER_MIN_TM=58 PRIMER_OPT_TM=62 PRIMER_MAX_TM=66 PRIMER_OPT_GC_PERCENT=60

What should I change? Thanks!

By the way, I am not sure why it doesn't show well but each block is finished by a '=' on a new line plus at least one empty new line

EDIT: Oops I got it, no new line between each block!