Result of running of p3_in.pl

Question

[nasri]

I have run the p3_in.pl script, but the sequence value is empty. The Illumina NGS data that I ran was MISA format data from the FLASH running results. This is the result:

"
PRIMER_SEQUENCE_ID=_1
SEQUENCE=
PRIMER_PRODUCT_SIZE_RANGE=100-280
TARGET=24,20
PRIMER_MAX_END_STABILITY=250
=
PRIMER_SEQUENCE_ID=_1
SEQUENCE=
PRIMER_PRODUCT_SIZE_RANGE=100-280
TARGET=46,20
PRIMER_MAX_END_STABILITY=250
=
............
"

Is it like that? Next, I tried running on Primer 3, but the results were only like this in a file size of 1 kb:

PRIMER_SEQUENCE_ID=_1
SEQUENCE=
PRIMER_PRODUCT_SIZE_RANGE=100-280
TARGET=24,20
PRIMER_MAX_END_STABILITY=250
PRIMER_ERROR=thermodynamic approach chosen, but path to thermodynamic parameters not specified
=

[GenoMax]

Please include the command lines you are running. Without them the errors/output have no context. What is MISA format data?

[nasri]

I run this command

perl p3_in.pl TN_sequence.misa

Output is:

" PRIMER_SEQUENCE_ID=_1 SEQUENCE= PRIMER_PRODUCT_SIZE_RANGE=100-280 TARGET=24,20

PRIMER_MAX_END_STABILITY=250
PRIMER_SEQUENCE_ID=_1 SEQUENCE= PRIMER_PRODUCT_SIZE_RANGE=100-280 TARGET=46,20

PRIMER_MAX_END_STABILITY=250
............

After that, I run the Primer 3 use command:

primer3_core -o TN_p3.p3out TN_misa_inp3.p3in

Output is:

PRIMER_SEQUENCE_ID=_1 SEQUENCE= PRIMER_PRODUCT_SIZE_RANGE=100-280 TARGET=24,20 PRIMER_MAX_END_STABILITY=250

PRIMER_ERROR=thermodynamic approach chosen, but path to thermodynamic parameters not specified.

The MISA format data, I mean that "TN_sequence.misa"